- Email: [email protected]
A novel FeS cluster in Fe-only hydrogenases
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27 Ulmasov, T. et al. (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971 28 Ulmasov, T. et al. (1997) ARF1, a transcription factor that binds to auxin response elements. Science 276, 1865–1868 29 Ulmasov, T. et al. (1999) Dimerization and DNA binding of auxin response factors. Plant J. 19, 309–319 30 Leyser, H.M.O. et al. (1996) Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J. 10, 403–413 31 Rouse, D. et al. (1998) Changes in auxin response
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from mutations in an Aux/IAA gene. Science 279, 1371–1373 Tian, Q. and Reed, J.W. (1999) Control of auxinregulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126, 711–721 Oeller, P.W. and Theologis, A. (1995) Induction kinetics of the nuclear proteins encoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 and PSIAA6, in pea (Pisum sativum L.). Plant J. 7, 37–48 Cernac, A.C. et al. (1997) The SAR1 gene of Arabidopsis acts downstream of the AXR1 gene in auxin response. Development 124, 1583–1591 Li, S.J. and Hochstrasser, M. (1999) A new protease required
for cell-cycle progression in yeast. Nature 398, 246–251 36 Jones, A.M. et al. (1998) Auxin-dependent cell expansion mediated by overexpressed Auxin-Binding Protein 1. Science 282, 1114–1117 37 Winston, J.T. et al. (1999) The SCFb-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkBa and bcatenin and stimulates IkBa ubiquitination in vitro. Genes Dev. 13, 270–283 38 Mizoguchi, T. et al. (1994) Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells. Plant J. 5, 111–122
Structural comparisons
A novel FeS cluster in Fe-only hydrogenases Yvain Nicolet, Brian J. Lemon, Juan C. Fontecilla-Camps and John W. Peters Many microorganisms can use molecular hydrogen as a source of electrons or generate it by reducing protons. These reactions are catalysed by metalloenzymes of two types: NiFe and Fe-only hydrogenases. Here, we review recent structural results concerning the latter, putting special emphasis on the characteristics of the active site. IN 1931, Stephenson and Stickland showed that the facultative anaerobic colon bacterium Escherichia coli could activate hydrogen thanks to enzymes they termed hydrogenases1. More recently, these enzymes have been shown to play a central role in the hydrogen metabolism of many microorganisms of great biotechnological interest, such as methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria. Knowledge of the functional and structural properties of hydrogenases might help in the design of less expensive catalysts for fuel cells. Existing fuel cells use platinum catalysts and these severely restrict the use of hydrogendriven vehicles in spite of the urgent need to switching to clean fuels2. Hydrogenases catalyse the reversible reaction H2 ↔ 2H1 1 2e2 (Refs 3–6). The first Y. Nicolet and J.C. Fontecilla-Camps are at the Laboratoire de Cristallographie et de Cristallogenèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel, CEACNRS, 41 Avenue des Martyrs, 38027, Grenoble Cedex 1, France; and B.J. Lemon and J.W. Peters are at the Dept of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA. Email: [email protected]; [email protected]
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step in the reaction is the heterolytic cleavage of H2 into H1 and H2. The hydrogenuptake reaction results in protons and electrons, which are subsequently used to generate ATP and reducing power; the hydrogen-producing reaction involves the reduction of protons by lowredox-potential electrons generated by fermentation. Most of the hydrogenases described to date are metalloproteins containing Ni or Fe, or both. Of these, the NiFe hydrogenases are the most extensively studied and the three-dimensional structures of several enzymes belonging to this group have been reported7. A surprising feature of these enzymes is that their active sites contain, in addition to a Ni ion, a redox-inactive, low-spin Fe21 center with CO and CN2 coordination8,9. The unrelated Fe-only hydrogenases have been less well studied and, until very recently, no three-dimensional structure was available for this class of enzyme. However, in the past year or so, the structures of the Fe-only hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum have been solved to 1.6 and 1.8 Å resolution, respectively10,11. Here, we compare these structures and discuss their analogies to the NiFe-containing enzymes.
Both amino acid sequence analyses and electronic paramagnetic resonance (EPR) studies have indicated that Feonly hydrogenases generally contain two [4Fe–4S] clusters (the F-clusters) in a ferredoxin-like domain. In addition, an unusual EPR signal has been attributed to a novel 6Fe cluster that was proposed to be the active site and was called the H-cluster5. The positions of the F- and Hclusters in the respective F- and Hdomains of C. pasteurianum hydrogenase I (CpI) and D. desulfuricans hydrogenase (DdH) are shown in Fig. 1. DdH is a dimeric periplasmic protein of 53 kDa (43 kDa and 10 kDa for the large and small subunits, respectively) and CpI is a monomeric cytoplasmic enzyme of 61 kDa. In addition to the clusters described above, CpI contains two FeS centers coordinated by domains found at the N terminus of the molecule. One of these domains bears a striking structural similarity to [2Fe–2S] plant-type ferrodoxins (green in Fig. 1b,c). A short domain (pink in Fig. 1b,c) connects the N-terminal [2Fe–2S] ferrodoxin-like module with the F-domain and consists of just two a helices separated by a loop region that coordinates an additional [4Fe–4S] cluster through three cysteinyl ligands and the Ne ring atom of a histidine residue. As was expected from the high degree of amino acid sequence identity (Fig. 1a)12,13, CpI and DdH display extensive structural similarities at the Hand F-domains (Fig. 1b,c)10,11. The rootmean-square deviation (rmsd) for the superposition between the H-domains is 1.0 Å (338 Ca’s) and that between the Fdomains is 1.7 Å (57 Ca’s). However, the relative orientations of the H- and Fdomains are not the same in the two enzymes: the rmsd for the combined Hand F-domains (381 Ca’s) is 2.2 Å. This is probably a consequence of differences elsewhere in the molecules, such as the two extra N-terminal domains in CpI, the numerous insertions in the F-domain in CpI and the longer C-terminal region of
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
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TIBS 25 – MARCH 2000 the small subunit in DdH10,11 (red in Fig. 1b,c). This subunit does not constitute a domain but rather a stretched polypeptide chain that completely embraces the large subunit (dark blue in Fig. 1b,c). Thus, in DdH, both the large and the small subunit belong to the Hdomain and are topologically equivalent to the single chain of CpI (Fig. 1c). Consequently, DdH’s large-subunit C terminus and small-subunit N terminus are very close in space. In CpI, the equivalent regions are connected by a loop (yellow in Fig. 1b,c).
(a) DdH_L CpI
.......................................................................................... MKTIIINGVQFNTDEDTTILKFARDNNIDISALCFLNNCNNDINKCEICTVEVEGTGLVTACDTLIEDGMIINTNSDAVNEKIKSRISQL 1
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IEA......CINCGQCLTHCPENAIYEAQSWVPEVEKKLKDGKVKCIAMPAPAVRYALGDAFGMPVGSVTTGKMLAALQKLGFAHCWDTE DEKCFDDTNCLLCGQCIIACPVAALSE.KSHMDRVKNALNAPEKHVIVAMAPSVRASIGELFNMGFGVDVTGKIYTALRQLGFDKIFDIN
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FTADVTIWEEGSEFVERLTKKSDMPLPQFTSCCPGWQKYAETYYPELLPHFSTCKSPIGMNGALAKTYGAERMKYDPKQVYTVSIMPCIA FGADMTIMEEATELVQRIE..NNGPFPMFTSCCPGWVRQAENYYPELLNNLSSAKSPQQIFGTASKTYYPSISGLDPKNVFTVTVMPCTS
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KKYEGLRPELKSSGMRDIDATLTTRELAYMIKKAGIDFAKLPDGKRDSLMGESTGGATIFGVTGGVMEAALRFAYEAVTGKKPDSWDFKA KKFEADRPQMEKDGLRDIDAVITTRELAKMIKDAKIPFAKLEDSEADPAMGEYSGAGAIFGATGGVMEAALRSAKDFAENAELEDIEYKQ 330
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......................MSRTVMERIEYEMHTPDPKADPDKLHFVQIDEAKCIGCDTCSQYCPTAAIFGEMGEP......HSIPH LDIHEFKCGPCNRRENCEFLKLVIKYKARASKPFLPKDKTEYVDERSKSLTVDRTKCLLCGRCVNACGKNTETYAMKFLNKNGKTIIGAE
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VRGLDGIKEATVNVGGTDVKVAVVHGAKRFKQVCDDVKAGKSPYHFIEYMACPGGCVCGGGQPVMPGVLEAMDRTTTRLYAGLKKRLAMA VRGLNGIKEAEVEINNNKYNVAVINGASNLFKFMKSGMINEKQYHFIEVMACHGGCVNGGGQPH..VNPKD...................
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MQIASITRRGFLKVACVTTGAALIGIRMTGKAVAAVKQIKDYMLDRINGVYGADAKFPVRASQDNTQVKALYKSYLEKPLGHKSHDLLHT ...................................LEK.VDIKKVRASVLYNQDEHLSKRKSHENTALVKMYQNYFGKPGEGRAHEILH.
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Periplasmic versus cytoplasmic hydrogenases The location of a hydrogenase in the bacterial cell should reflect the enzyme’s function. DdH is a periplasmic protein whose physiological role is hydrogen uptake: in Desulfovibrio sp., in general, protons resulting from the oxidation of hydrogen by the Fe hydrogenase in the periplasm create a gradient across the membrane that is thought to be coupled to ATP synthesis in the cytoplasm. The resulting electrons are also transferred to the cytoplasm by a redox transmembrane complex14, where they are used in a stepwise manner to reduce sulfate to sulfide or to generate reducing power for the cell. CpI is a cytoplasmic, hydrogen-producing enzyme. In fermenting Clostridia sp., ferredoxin transfers two electrons to CpI, which, in turn, uses protons as electron acceptors to generate hydrogen. This reaction rids the cell of an excess of low-potential electrons and regenerates oxidized ferredoxin. It has been shown that protein translocation to the periplasm requires a specific signal peptide that includes the consensus sequence RRxFxK and is subsequently cleaved to form the mature, functional molecule15. In periplasmic Fe hydrogenases, the gene sequence corresponding to the 34amino-acid signal peptide is attached to the N terminus of the small subunit within the H-domain (Fig. 1b, dashed line). In addition to the gene sequence corresponding to the signal peptide, another sequence that putatively codes for 24 amino acids is found beyond the C-terminal end of the large subunit10,13. The polypeptide corresponding to this gene sequence (Fig. 1b, dashed line) is not present in the purified enzyme; it might represent an additional signal peptide involved in translocation or localization, or both, of the large subunit to the periplasm.
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Figure 1 (a) Amino acid sequences of Desulfovibrio desulfuricans Fe hydrogenase (DdH) and Clostridium pasteurianum Fe-hydrogenase I (CpI), deduced from the gene sequences, aligned to take into account the three-dimensional structure homology. ■, ▲ and ● indicate the cysteines involved in the distal, medial and proximal FeS clusters (from the active site), respectively] DdH_L and DdH_S refer to the large and small subunit of DdH, respectively. The first 34 residues deduced from the gene sequence of DdH_S are missing in the native protein. This fragment is known to be a signal peptide necessary for protein export to the periplasmic space and it is subsequently removed. Also, the last 24 residues of DdH_L deduced from the gene sequence have no equivalent in CpI and are also absent in purified DdH. (b) Schematic view of the various domains of DdH and CpI. Structurally related domains are depicted in the same color: the [2Fe–2S] plant-ferredoxin-like domain (Fd) is green; the atypical [4Fe–4S]cluster-containing small domain is pink; the F-domain (2[4Fe–4S] ferredoxin-like domain) is cyan. The large- and small-subunit parts of the H-domain in DdH, and their homologous parts in CpI, are represented in dark blue and red, respectively. The loop connecting these two parts in CpI is yellow, and the dotted line indicates a gap in the sequence alignment arising from peptide insertions at the N- and C-terminal ends of DdH_S and DdH_L, respectively. (c) Threedimensional structures of DdH (left) and CpI (right). The colors used are the same as in (b). In DdH, the small subunit (red) completely embraces the large one (dark blue). The C terminus of the large subunit is very close to the N terminus of the small subunit. There is also a buried non-protein cysteine molecule that links these two regions. In the monomeric CpI, the equivalent region is continuous (yellow). The FeS clusters and the cysteine ligand are represented by spheres.
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TIBS 25 – MARCH 2000 are structural differences between CpI and DdH in this region, the respective pockets have different solvent accessibilities (Fig. 3). In DdH, the cavity is blocked from solvent by the smallsubunit C-terminal region, the Nterminal helix of the small subunit (which has one extra turn relative to CpI) and the C-terminal region of the large subunit. In CpI, the position of the [8Fe–8S] ferredoxin-like domain generates a network of water molecules running from the solvent to the cavity.
Active site Figure 2 (a) A difference Fourier-transform electron-density map of Desulfovibrio desulfuricans Fe hydrogenase (DdH) calculated from a model in which the nonprotein cysteine was removed from the phase and structure-factors calculations (an omit map). The dotted lines represent the hydrogen bonds between the protein and the cysteine molecule. The second letter (L or S) in the name of the residues represents the large or small subunit, respectively. (b) The homologous region in Clostridium pasteurianum Fe-hydrogenase (CpI), shown in the same orientation. The omit map shows the positions of four internal water molecules. This figure was prepared using MOLSCRIPT26, MINIMAGE27 and RASTER3D28. (a)
(b) N terminus small subunit
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Figure 3 Accessibility surfaces of (a) Desulfovibrio desulfuricans Fe hydrogenase (DdH) and (b) Clostridium pasteurianum Fe-hydrogenase I (CpI). The colors are as in Fig. 1. The region described in Fig. 2 is clearly accessible to solvent in CpI, whereas the longer C terminus of the small subunit (red) in DdH covers this region. This completely blocks the cysteine-containing cavity in DdH, which is thus not accessible to the solvent medium. The surfaces were calculated with GRASP29 using a PDB file with internal water molecules removed and a probe radius of 1.4 Å.
The monomeric cytoplasmic hydrogenases are likely to be ancestral to the heterodimeric periplasmic ones because of the unusual topology of the small subunit in the latter (Fig. 1c). Translocation to the periplasm required gene splitting and signal-peptide insertions in the ancestral monomeric cytoplasmic enzyme.
Cysteine versus water molecules In DdH, a putative free cysteine amino acid is found in an internal pocket close
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to the protein surface, where it establishes hydrogen bonds with several residues, thus connecting the C and N termini of the large and small subunits (Fig. 2). Binding of this small molecule probably became necessary for the stabilization of these regions after the putative gene-splitting event in DdH that generated the small and large subunit. Accordingly, it is absent from the monomeric CpI, in which the corresponding pocket is occupied by four water molecules (Fig. 2b). Because there
The determination of the structure of the active site of Fe-only hydrogenases has revealed a new biological FeS cluster (Fig. 4). One of the unusual features of the active-site Fe binuclear center of both CpI and DdH structures is the presence of two terminal nonprotein ligands at each Fe atom. In the crystallographic analyses, it was assumed that these ligands represented CN2 or CO, or both, because of similarities between the Fourier-transform infrared (FTIR) spectra of Fe-only and NiFe hydrogenases9. [As mentioned above, studies involving X-ray diffraction and FTIR spectroscopy have established that the Fe site of NiFe hydrogenases contains one CO and two CN2 (Fig. 5c).] These observations have recently been confirmed by a study on the Fe-only hydrogenase from Desulfovibrio vulgaris, which indicated that this enzyme should contain at least one CO and one CN2 as terminal ligands to an Fe atom16. Because X-ray analysis cannot discriminate between CO and CN2, thanks to the small difference in the number of electrons of these two moieties, their assignment must rely on indirect evidence. One important aspect is the protein environment: the charged CN2 ligands are more likely to form hydrogen bonds than to sit on hydrophobic pockets; CO ligands can probably do both. In the active sites of CpI and DdH, hydrogen bonding at the diatomic moiety on the Fe atom distal to the [4Fe–4S] cluster (Fe2) is provided by the Ne of K358 and K237, respectively. Consequently, this diatomic ligand has been tentatively assigned as CN2 (Figs 4 and 5a,b). In the CpI structure, one diatomic moiety on the Fe atom next to the [4Fe–4S] cluster (Fe1) is hydrogen bonded to the Og of S232. Interestingly, this residue is not conserved in the DdH enzyme, in which the equivalent residue is A109. However, hydrogen bonding occurs at this position through the peptide-bond amide of the alanine.
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TIBS 25 – MARCH 2000 Differences in hydrogen bonding to the putative cyanide ligands might be important in dictating the midpoint potential of the binuclear Fe center and this is one of the factors that should determine the direction of the reaction (either hydrogen oxidation or hydrogen production). Another determining factor could be the local hydrogen concentration. The other two terminal diatomic ligands occupy hydrophobic pockets and have been modeled as CO. The presence of hydrogen-bond donors in close proximity to two of the diatomic ligands of the Fe site of the NiFe hydrogenase was the basis for the tentative assignment of CN2 rather than CO in that structure8. The active site Fe1 and Fe2 are bridged by two S atoms that are located 3.0 Å apart and are connected by a moiety of undetermined identity (Fig. 4). This moiety was originally identified in the CpI structure as a water molecule located ~2.7 Å from each S atom. However, the considerable residual electron density in difference electron density maps (using experimental against calculated diffraction data) suggests that the moiety consists either of a heavier atom at this position or of multiple atoms that covalently link the two S atoms. For the 1.6-Å-resolution structure of DdH, the electron-density map clearly indicates three covalently bound light atoms bridging the two sulfur atoms. The resulting small molecule has been modelled as a 1,3 propanedithiolate (PDT). It should be pointed out, however, that it is not possible to distinguish unambiguously between C, N and O, and so a combination of light atoms other than the three carbons proposed in PDT cannot be ruled out. This is especially true for the central atom, which makes close contact with the Sg of C178 in DdH. An equivalent three-atom linkage would be consistent with the data observed in the CpI structure, and it is probable that the linkages in both enzymes are the same. A covalent connection between the two S atoms is bound to have an impact on the electronic properties and the net charge of the H cluster. A feature that is reportedly different between the two structures is the identity of the active-site-Fe-bridging organic ligand. In CpI, the crystallographic refinement clearly indicates that it is a diatomic ligand, presumably CO, but the electron density in DdH is best modeled as an asymmetrically coordinated water molecule. However, the observed Fe-to‘water’ distance of 2.6 Å in DdH argues against an aqueous ligand and favors
Figure 4 Comparison of selected amino acid residues in the environment of the 2Fe subcluster of the H cluster of (a) Clostridium pasteurianum Fe-hydrogenase I (CpI) and (b) Desulfovibrio desulfuricans Fe hydrogenase (DdH). The [4Fe–4S] subcluster and associated Cys ligands are included to provide the proper perspective and are shown in gray. Fe atoms of the 2Fe subcluster, large red spheres; sulfur, yellow; oxygen, small red spheres; nitrogen, blue; carbon, white. A question mark indicates the unassigned moiety that bridges two sulfur atoms in CpI; a propane linkage has been putatively assigned at the analogous position in DdH. This figure was prepared with MOLSCRIPT26, MINIMAGE27 and RASTER3D28.
Figure 5 Comparison of the active site 2Fe subclusters of (a) Clostridium pasteurianum Fe-hydrogenase I (CpI), (b) Desulfovibrio desulfuricans Fe hydrogenase (DdH) and (c) the bimetallic active site of the Desulfovibrio gigas NiFe hydrogenase. The color scheme is the same as that shown in Fig. 4, except that the S bridging moiety in CpI is represented as a magenta sphere. This figure was prepared with MOLSCRIPT26, MINIMAGE27 and RASTER3D28.
a partially disordered, asymmetrically bound, diatomic ligand instead. FTIR studies of the D. vulgaris Fe-only hydrogenase suggest that a bridging CO is present in the oxidized state but absent when the enzyme is reduced16. Because the crystalline DdH is likely to be more reduced than the equivalent CpI (see below), the differences between the two active sites, as far as the bridging ligand is concerned, could simply reflect variations in their redox states. Thus, neither the X-ray analysis nor the FTIR studies can eliminate the possibility that the bridging ligand could alternate between an Fe-to-Fe bridging and an Fe terminally bound state during turnover. These questions, as well as the details of the chemical mechanism, will only be answered by additional biochemical and structural studies of the individual oxidation states.
Catalytic mechanism The details of the structure of the Fe binuclear center of the H cluster from both CpI and DdH suggest possible mechanisms for reversible hydrogen oxidation at the site. This center is remarkably similar in the two structures, but there are several subtle differences that are important to the mechanism. As suggested above, the main differences in the two structures are probably attributable to the oxidation state. Although it has not been within our means to characterize the oxidation state of the hydrogenase crystals directly, the CpI active site structure probably represents an anaerobically oxidized state and the D. desulfuricans structure a more-reduced state. This seems to be the most likely scenario for two reasons: first, crystals were grown under different conditions (CpI under
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Substrate access to the active site There is a continuous hydrophobic channel running from the molecule’s surface to the active site, where it points at the apparently vacant Fe2 coordination site in the DdH structure10. This channel is also present in the CpI enzyme. Furthermore, the amino acids that line the channel are strictly conserved in the two Fe hydrogenases, suggesting that the same internal pathway is used for either the uptake or the production of molecular hydrogen. The channel is analogous to those observed in the Desulfovibrio fructosovorans NiFe hydrogenase. These channels have been shown to be likely to facilitate hydrogen access to the active site in this class of enzyme18.
Proton transfer The reaction intermediates of the production or uptake of hydrogen by hydrogenases are protons and hydrides3–6. These species result either from the transport of protons and their reduction by the buried active site (production) or from the heterolytic cleavage of the hydrogen molecule (uptake). In both instances, it is necessary to postulate the existence of proton-transfer pathways either to or from the solvent medium because the active sites are buried in the protein core. The CpI and DdH structures reveal several potential proton donor and acceptor groups near the active site of the two Fe hydrogenases10,11. The most obvious possibilities are conserved in both structures and
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TIBS 25 – MARCH 2000 include the following: (1) A lysine residue (K358 in CpI, K237 in DdH) that, as indicated above, forms a hydrogen bond with one of the putative CN2 ligands and is at ~4 Å from Fe2 (the putative hydrogen-binding/protonreduction site, see above). (2) A cysteine thiol (C299 in CpI, C178 in DdH) is ~5.0 Å from Fe2 and, in CpI, is within hydrogen-bond distance of the terminally bound water molecule coordinated at this site. (3) The bridging thiolates of the 2Fe cluster, for which protonation is not energetically favorable19 and might disrupt the binuclear Fe center, so their role as proton donors or acceptors is unlikely. The lysine residue mentioned above is the closest potential donor–acceptor molecule to Fe2. If K237 is indeed the catalytic base that assists in the heterolytic cleavage of molecular hydrogen, then a plausible proton-transfer pathway exists in DdH involving E240, three water molecules and E245 at the molecule’s surface. The involvement of this lysine residue as a general acid–base would modify the hydrogenbonding structure of the putative CN2 ligand of Fe2. It might be possible to detect such modification using FTIR for well-defined redox states. In CpI, a water molecule is close (~3.5 Å) to a free cysteine residue (C299) that lies ~5.0 Å from Fe2. It seems reasonable that hydride formation could occur by the displacement of the bound water molecule at this site, with the cysteine-bound water molecule acting as a proton donor– acceptor. The loss of the coordination of the water molecule by the distal Fe atom is consistent with the DdH structure, which reveals an apparent open coordination site at this position. A potential problem is the van der Waals interaction between the S of this cysteine and the central atom of the putative PDT (d 5 3.2 Å; d, distance). However, as mentioned above, if the latter atom is not C but either O or N, the observed distance would not be a problem. Thus, the cysteine residue is an attractive prospect as a proton donor–acceptor with a pKa of ~8.0 and strict conservation among all Fe-only hydrogenases for which the primary sequence has been determined12,13,20–25. Additional experimental investigation will be required to assign the proton donor–acceptor(s) definitively in the Fe-only hydrogenases.
Conclusions In spite of the fact that Fe-only hydrogenases from fermenting and
sulfate-reducing bacteria catalyse opposed reactions, they display many common features: the F- and H-domains have very similar foldings, and the active sites are almost identical. Major differences seem to derive from the localization of the enzymes. In DdH, a genesplitting event has resulted in the addition of one, or perhaps two, signal sequences that are needed for translocation to the periplasm. CpI has two extra domains at its N-terminal region that coordinate FeS clusters and may be instrumental in the interactions of the hydrogenase with redox partners. Residues surrounding the active site suggest ways through which protons can arrive at or departure from the active site. One of the Fe atoms of the binuclear center (Fe2) seems to be an excellent candidate for the initial substrate-binding site. A comparison between the active sites of NiFe and Fe-only hydrogenases (Fig. 5) strongly suggests that a low-spin Fe(II) with CO and CN2 coordination is central to hydrogen metabolism, perhaps as a hydride-binding species.
References 1 Stephenson, M. and Stickland, L.H. (1931) Hydrogenase: a bacterial enzyme activating molecular hydrogen. I. The properties of the enzyme. Biochem. J. 25, 205–214 2 Berger, D.J. (1999) Fuel cells and precious-metal catalysis. Science 286, 49 3 Schlegel, H.G. and Schneider K. (1978) In Hydrogenases (Schlegel, H.G. and Schneider, K., eds), pp. 15–44, Erich Goltze KG, Göttingen, Germany 4 Adams, M.W. et al. (1980) Hydrogenase. Biochim. Biophys. Acta 594, 105–176 5 Adams, M.W. (1990) The structure and mechanism of iron-hydrogenases. Biochim. Biophys. Acta 1020, 115–145 6 Albracht, S.P. (1994) Nickel hydrogenases: in search of the active site. Biochim. Biophys. Acta 1188, 167–204 7 Volbeda, A. et al. (1995) Crystal structure of the nickeliron hydrogenase from Desulfovibrio gigas. Nature 373, 580–587 8 Volbeda, A. et al. (1996) Structure of the [NiFe] hydrogenase active site: evidence for biologically uncommon Fe ligands. J. Am. Chem. Soc. 118, 12989–12996 9 Happe, R.P. et al. (1997) Biological activation of hydrogen. Nature 385, 126 10 Nicolet, Y. et al. (1999) Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Struct. Fold. Des. 7, 13–23 11 Peters, J.W. et al. (1998) X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853–1858 (Errata: 283, 35; 283, 2102) 12 Meyer, J. and Gagnon, J. (1991) Primary structure of hydrogenase I from Clostridium pasteurianum. Biochemistry 30, 9697–9704 13 Hatchikian, E.C. et al. (1999) Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757. J. Bacteriol. 181, 2947–2952 14 Pereira, I.A.C. et al. (1998) Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfuvibrio sp. J. Biol. Inorg. Chem. 3, 494–498 15 Nivière, V. et al. (1992) Site-directed mutagenesis of the hydrogenase signal-peptide consensus box prevents export of a b-lactamase fusion protein. J. Gen. Microbiol. 138, 2173–2183 16 Pierik, A.J. et al. (1998) A low-spin iron with CN and CO as intrinsic ligands forms the core of the active site in [Fe]-hydrogenases. Eur. J. Biochem. 258, 572–578
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TIBS 25 – MARCH 2000 17 Lemon, B.J. and Peters, J.W. (1999) Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum. Biochemistry 38, 12969–12973 18 Montet, Y. et al. (1997) Gas access to the active site of Ni–Fe hydrogenases probed by X-ray crystallography and molecular dynamics. Nat. Struct. Biol. 4, 523–526 19 Niu, S. et al. (1999) Theoretical characterization of the reaction intermediates in a model of the nickel–iron hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. 121, 4000–4007 20 Gorwa, M.F. et al. (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J. Bacteriol. 178, 2668–2675
21 Malki, S. et al. (1995) Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans. J. Bacteriol. 177, 2628–2636 22 Stokkermans, J. et al. (1989) hyd gamma, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase. FEMS Microbiol. Lett. 49, 217–222 23 Voordouw, G. et al. (1985) Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH2-terminal sequence. Eur. J. Biochem. 148. 509–514 24 Voordouw, G. et al. (1989) Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello. J. Bacteriol. 171, 3881–3889
Eukaryotic DNA polymerases, a growing family Ulrich Hübscher, Heinz-Peter Nasheuer and Juhani E. Syväoja In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called a, b, g, d, e, z, h, u and i), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase. ANY LIVING CELL and organism is faced with the tremendous task of keeping the genome intact in order to develop in an organized manner, function in a complex environment, divide at the right time and die when it is appropriate. To achieve this, DNA synthesis is required to duplicate the genetic information prior to cell division. DNA synthesis is also needed during DNA repair processes, including DNA recombination and bypassing lesions when the DNA has been damaged (translesion DNA synthesis). DNA synthesis is performed by enzymes, called DNA polymerases (DNA pol)1. Since the U. Hübscher is at the Institute of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; H-P. Nasheuer is at the Institute of Molecular Biotechnology, Dept of Biochemistry, Beutenbergstrasse 11, D-07745 Jena, Germany; and J.E. Syväoja is at the Biocenter Oulu and Dept of Biochemistry, University of Oulu, FIN-90570 Oulu, Finland, and Dept of Biology, University of Joensuu, FIN-80100 Joensuu, Finland. Email: [email protected]
discovery of DNA pol a in eukaryotic cells in 1957, the number of DNA pols identified has grown. In the early 1970s, DNA pol b and g were discovered leading to the simple concept that DNA pol a is the enzyme involved in DNA replication, DNA pol b in DNA repair and DNA pol g in mitochondrial DNA replication. However, the discovery of DNA pol d and e in mammalian cells during the 1980s complicated this interpretation. It also suggested that a particular DNA pol might have more than one functional task in a cell, and that a particular DNA synthetic event can require more than one DNA pol (reviewed in Ref. 2). Genetic studies performed with budding yeast Saccharomyces cerevisiae, for example, showed that the three DNA pols a, d and e share the task of replicating the cellular genome, and that DNA repair events such as base excision repair might require not only DNA pol b but also DNA pol d or DNA pol e, or both, especially in longpatch base excision repair. Because both replication and repair are of primary importance for cells and organisms, it
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25 Santangelo, J.D. et al. (1995) Characterization and expression of the hydrogenase-encoding gene from Clostridium acetobutylicum P262. Microbiology 141, 171–180 26 Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 27 Arnez, J.G. (1994) MINIMAGE: a program for plotting electron-density maps. J. Appl. Crystallogr. 27, 649–653 28 Merritt, E.A. and Bacon, D.J. (1997) Raster3D photorealistic molecular graphics. Methods Enzymol. 277, 505–524 29 Nicholls, A. (1992) GRASP: Graphical Representation and Analysis of Surface Properties, Colombia University
appears that nature created safety mechanisms by employing various DNA pols for similar functional tasks. Translesion DNA synthesis, for example, requires at least DNA pols z and h, the former probably being responsible for error-prone translesion DNA synthesis and the latter performing error-free DNA translesion synthesis (reviewed in Ref. 3). In many cases, DNA pols have complex polypeptide structures because, in addition to the polymerizing subunit (that often contains a proofreading 39→59 exonuclease), they comprise other functional subunits (Table 1). These functions include other enzymatic activities (e.g. DNA primase) or allow the DNA polymerase to interact with other proteins, involved in check-point function, cell cycle control, DNA replication or DNA repair. The replicative DNA pols d and e, for example, are chaperoned by the accessory proteins replication factor C (RF-C), and proliferating cell nuclear antigen (PCNA). The interaction of these two accessory proteins with the DNA pols permits a high speed of DNA synthesis coupled with a high accuracy4. At the structural level, DNA pols appear to possess a universal DNA polymerase active site5. This is achieved by a two-metal-ion-catalysed mechanism and guarantees the incorporation of the correctly base-paired deoxyribonucleoside triphosphate (according to the Watson– Crick base pairing rule A–T and G–C) onto a growing template. However, DNA pols do differ in various aspects of their structural architecture5, as a result of the many possible interactions of DNA pols with other proteins and enzymes. Thus, the active site of the DNA pol is very conserved in evolution, whereas the structure of the surface of the molecules might differ considerably.
Functional roles of DNA pol a
DNA polymerase a–primase (DNA pol a– prim) has an important role in DNA replication (Fig. 1, reviewed in Ref. 6).
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27 Ulmasov, T. et al. (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971 28 Ulmasov, T. et al. (1997) ARF1, a transcription factor that binds to auxin response elements. Science 276, 1865–1868 29 Ulmasov, T. et al. (1999) Dimerization and DNA binding of auxin response factors. Plant J. 19, 309–319 30 Leyser, H.M.O. et al. (1996) Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J. 10, 403–413 31 Rouse, D. et al. (1998) Changes in auxin response
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from mutations in an Aux/IAA gene. Science 279, 1371–1373 Tian, Q. and Reed, J.W. (1999) Control of auxinregulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126, 711–721 Oeller, P.W. and Theologis, A. (1995) Induction kinetics of the nuclear proteins encoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 and PSIAA6, in pea (Pisum sativum L.). Plant J. 7, 37–48 Cernac, A.C. et al. (1997) The SAR1 gene of Arabidopsis acts downstream of the AXR1 gene in auxin response. Development 124, 1583–1591 Li, S.J. and Hochstrasser, M. (1999) A new protease required
for cell-cycle progression in yeast. Nature 398, 246–251 36 Jones, A.M. et al. (1998) Auxin-dependent cell expansion mediated by overexpressed Auxin-Binding Protein 1. Science 282, 1114–1117 37 Winston, J.T. et al. (1999) The SCFb-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkBa and bcatenin and stimulates IkBa ubiquitination in vitro. Genes Dev. 13, 270–283 38 Mizoguchi, T. et al. (1994) Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells. Plant J. 5, 111–122
Structural comparisons
A novel FeS cluster in Fe-only hydrogenases Yvain Nicolet, Brian J. Lemon, Juan C. Fontecilla-Camps and John W. Peters Many microorganisms can use molecular hydrogen as a source of electrons or generate it by reducing protons. These reactions are catalysed by metalloenzymes of two types: NiFe and Fe-only hydrogenases. Here, we review recent structural results concerning the latter, putting special emphasis on the characteristics of the active site. IN 1931, Stephenson and Stickland showed that the facultative anaerobic colon bacterium Escherichia coli could activate hydrogen thanks to enzymes they termed hydrogenases1. More recently, these enzymes have been shown to play a central role in the hydrogen metabolism of many microorganisms of great biotechnological interest, such as methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria. Knowledge of the functional and structural properties of hydrogenases might help in the design of less expensive catalysts for fuel cells. Existing fuel cells use platinum catalysts and these severely restrict the use of hydrogendriven vehicles in spite of the urgent need to switching to clean fuels2. Hydrogenases catalyse the reversible reaction H2 ↔ 2H1 1 2e2 (Refs 3–6). The first Y. Nicolet and J.C. Fontecilla-Camps are at the Laboratoire de Cristallographie et de Cristallogenèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel, CEACNRS, 41 Avenue des Martyrs, 38027, Grenoble Cedex 1, France; and B.J. Lemon and J.W. Peters are at the Dept of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA. Email: [email protected]; [email protected]
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step in the reaction is the heterolytic cleavage of H2 into H1 and H2. The hydrogenuptake reaction results in protons and electrons, which are subsequently used to generate ATP and reducing power; the hydrogen-producing reaction involves the reduction of protons by lowredox-potential electrons generated by fermentation. Most of the hydrogenases described to date are metalloproteins containing Ni or Fe, or both. Of these, the NiFe hydrogenases are the most extensively studied and the three-dimensional structures of several enzymes belonging to this group have been reported7. A surprising feature of these enzymes is that their active sites contain, in addition to a Ni ion, a redox-inactive, low-spin Fe21 center with CO and CN2 coordination8,9. The unrelated Fe-only hydrogenases have been less well studied and, until very recently, no three-dimensional structure was available for this class of enzyme. However, in the past year or so, the structures of the Fe-only hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum have been solved to 1.6 and 1.8 Å resolution, respectively10,11. Here, we compare these structures and discuss their analogies to the NiFe-containing enzymes.
Both amino acid sequence analyses and electronic paramagnetic resonance (EPR) studies have indicated that Feonly hydrogenases generally contain two [4Fe–4S] clusters (the F-clusters) in a ferredoxin-like domain. In addition, an unusual EPR signal has been attributed to a novel 6Fe cluster that was proposed to be the active site and was called the H-cluster5. The positions of the F- and Hclusters in the respective F- and Hdomains of C. pasteurianum hydrogenase I (CpI) and D. desulfuricans hydrogenase (DdH) are shown in Fig. 1. DdH is a dimeric periplasmic protein of 53 kDa (43 kDa and 10 kDa for the large and small subunits, respectively) and CpI is a monomeric cytoplasmic enzyme of 61 kDa. In addition to the clusters described above, CpI contains two FeS centers coordinated by domains found at the N terminus of the molecule. One of these domains bears a striking structural similarity to [2Fe–2S] plant-type ferrodoxins (green in Fig. 1b,c). A short domain (pink in Fig. 1b,c) connects the N-terminal [2Fe–2S] ferrodoxin-like module with the F-domain and consists of just two a helices separated by a loop region that coordinates an additional [4Fe–4S] cluster through three cysteinyl ligands and the Ne ring atom of a histidine residue. As was expected from the high degree of amino acid sequence identity (Fig. 1a)12,13, CpI and DdH display extensive structural similarities at the Hand F-domains (Fig. 1b,c)10,11. The rootmean-square deviation (rmsd) for the superposition between the H-domains is 1.0 Å (338 Ca’s) and that between the Fdomains is 1.7 Å (57 Ca’s). However, the relative orientations of the H- and Fdomains are not the same in the two enzymes: the rmsd for the combined Hand F-domains (381 Ca’s) is 2.2 Å. This is probably a consequence of differences elsewhere in the molecules, such as the two extra N-terminal domains in CpI, the numerous insertions in the F-domain in CpI and the longer C-terminal region of
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TIBS 25 – MARCH 2000 the small subunit in DdH10,11 (red in Fig. 1b,c). This subunit does not constitute a domain but rather a stretched polypeptide chain that completely embraces the large subunit (dark blue in Fig. 1b,c). Thus, in DdH, both the large and the small subunit belong to the Hdomain and are topologically equivalent to the single chain of CpI (Fig. 1c). Consequently, DdH’s large-subunit C terminus and small-subunit N terminus are very close in space. In CpI, the equivalent regions are connected by a loop (yellow in Fig. 1b,c).
(a) DdH_L CpI
.......................................................................................... MKTIIINGVQFNTDEDTTILKFARDNNIDISALCFLNNCNNDINKCEICTVEVEGTGLVTACDTLIEDGMIINTNSDAVNEKIKSRISQL 1
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IEA......CINCGQCLTHCPENAIYEAQSWVPEVEKKLKDGKVKCIAMPAPAVRYALGDAFGMPVGSVTTGKMLAALQKLGFAHCWDTE DEKCFDDTNCLLCGQCIIACPVAALSE.KSHMDRVKNALNAPEKHVIVAMAPSVRASIGELFNMGFGVDVTGKIYTALRQLGFDKIFDIN
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FTADVTIWEEGSEFVERLTKKSDMPLPQFTSCCPGWQKYAETYYPELLPHFSTCKSPIGMNGALAKTYGAERMKYDPKQVYTVSIMPCIA FGADMTIMEEATELVQRIE..NNGPFPMFTSCCPGWVRQAENYYPELLNNLSSAKSPQQIFGTASKTYYPSISGLDPKNVFTVTVMPCTS
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KKYEGLRPELKSSGMRDIDATLTTRELAYMIKKAGIDFAKLPDGKRDSLMGESTGGATIFGVTGGVMEAALRFAYEAVTGKKPDSWDFKA KKFEADRPQMEKDGLRDIDAVITTRELAKMIKDAKIPFAKLEDSEADPAMGEYSGAGAIFGATGGVMEAALRSAKDFAENAELEDIEYKQ 330
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......................MSRTVMERIEYEMHTPDPKADPDKLHFVQIDEAKCIGCDTCSQYCPTAAIFGEMGEP......HSIPH LDIHEFKCGPCNRRENCEFLKLVIKYKARASKPFLPKDKTEYVDERSKSLTVDRTKCLLCGRCVNACGKNTETYAMKFLNKNGKTIIGAE
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VRGLDGIKEATVNVGGTDVKVAVVHGAKRFKQVCDDVKAGKSPYHFIEYMACPGGCVCGGGQPVMPGVLEAMDRTTTRLYAGLKKRLAMA VRGLNGIKEAEVEINNNKYNVAVINGASNLFKFMKSGMINEKQYHFIEVMACHGGCVNGGGQPH..VNPKD...................
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MQIASITRRGFLKVACVTTGAALIGIRMTGKAVAAVKQIKDYMLDRINGVYGADAKFPVRASQDNTQVKALYKSYLEKPLGHKSHDLLHT ...................................LEK.VDIKKVRASVLYNQDEHLSKRKSHENTALVKMYQNYFGKPGEGRAHEILH.
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Periplasmic versus cytoplasmic hydrogenases The location of a hydrogenase in the bacterial cell should reflect the enzyme’s function. DdH is a periplasmic protein whose physiological role is hydrogen uptake: in Desulfovibrio sp., in general, protons resulting from the oxidation of hydrogen by the Fe hydrogenase in the periplasm create a gradient across the membrane that is thought to be coupled to ATP synthesis in the cytoplasm. The resulting electrons are also transferred to the cytoplasm by a redox transmembrane complex14, where they are used in a stepwise manner to reduce sulfate to sulfide or to generate reducing power for the cell. CpI is a cytoplasmic, hydrogen-producing enzyme. In fermenting Clostridia sp., ferredoxin transfers two electrons to CpI, which, in turn, uses protons as electron acceptors to generate hydrogen. This reaction rids the cell of an excess of low-potential electrons and regenerates oxidized ferredoxin. It has been shown that protein translocation to the periplasm requires a specific signal peptide that includes the consensus sequence RRxFxK and is subsequently cleaved to form the mature, functional molecule15. In periplasmic Fe hydrogenases, the gene sequence corresponding to the 34amino-acid signal peptide is attached to the N terminus of the small subunit within the H-domain (Fig. 1b, dashed line). In addition to the gene sequence corresponding to the signal peptide, another sequence that putatively codes for 24 amino acids is found beyond the C-terminal end of the large subunit10,13. The polypeptide corresponding to this gene sequence (Fig. 1b, dashed line) is not present in the purified enzyme; it might represent an additional signal peptide involved in translocation or localization, or both, of the large subunit to the periplasm.
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HWFDKSKGVKELTTAGKLPNPRASEFEGPYPYE ..FKYKK..........................
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Figure 1 (a) Amino acid sequences of Desulfovibrio desulfuricans Fe hydrogenase (DdH) and Clostridium pasteurianum Fe-hydrogenase I (CpI), deduced from the gene sequences, aligned to take into account the three-dimensional structure homology. ■, ▲ and ● indicate the cysteines involved in the distal, medial and proximal FeS clusters (from the active site), respectively] DdH_L and DdH_S refer to the large and small subunit of DdH, respectively. The first 34 residues deduced from the gene sequence of DdH_S are missing in the native protein. This fragment is known to be a signal peptide necessary for protein export to the periplasmic space and it is subsequently removed. Also, the last 24 residues of DdH_L deduced from the gene sequence have no equivalent in CpI and are also absent in purified DdH. (b) Schematic view of the various domains of DdH and CpI. Structurally related domains are depicted in the same color: the [2Fe–2S] plant-ferredoxin-like domain (Fd) is green; the atypical [4Fe–4S]cluster-containing small domain is pink; the F-domain (2[4Fe–4S] ferredoxin-like domain) is cyan. The large- and small-subunit parts of the H-domain in DdH, and their homologous parts in CpI, are represented in dark blue and red, respectively. The loop connecting these two parts in CpI is yellow, and the dotted line indicates a gap in the sequence alignment arising from peptide insertions at the N- and C-terminal ends of DdH_S and DdH_L, respectively. (c) Threedimensional structures of DdH (left) and CpI (right). The colors used are the same as in (b). In DdH, the small subunit (red) completely embraces the large one (dark blue). The C terminus of the large subunit is very close to the N terminus of the small subunit. There is also a buried non-protein cysteine molecule that links these two regions. In the monomeric CpI, the equivalent region is continuous (yellow). The FeS clusters and the cysteine ligand are represented by spheres.
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TIBS 25 – MARCH 2000 are structural differences between CpI and DdH in this region, the respective pockets have different solvent accessibilities (Fig. 3). In DdH, the cavity is blocked from solvent by the smallsubunit C-terminal region, the Nterminal helix of the small subunit (which has one extra turn relative to CpI) and the C-terminal region of the large subunit. In CpI, the position of the [8Fe–8S] ferredoxin-like domain generates a network of water molecules running from the solvent to the cavity.
Active site Figure 2 (a) A difference Fourier-transform electron-density map of Desulfovibrio desulfuricans Fe hydrogenase (DdH) calculated from a model in which the nonprotein cysteine was removed from the phase and structure-factors calculations (an omit map). The dotted lines represent the hydrogen bonds between the protein and the cysteine molecule. The second letter (L or S) in the name of the residues represents the large or small subunit, respectively. (b) The homologous region in Clostridium pasteurianum Fe-hydrogenase (CpI), shown in the same orientation. The omit map shows the positions of four internal water molecules. This figure was prepared using MOLSCRIPT26, MINIMAGE27 and RASTER3D28. (a)
(b) N terminus small subunit
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Figure 3 Accessibility surfaces of (a) Desulfovibrio desulfuricans Fe hydrogenase (DdH) and (b) Clostridium pasteurianum Fe-hydrogenase I (CpI). The colors are as in Fig. 1. The region described in Fig. 2 is clearly accessible to solvent in CpI, whereas the longer C terminus of the small subunit (red) in DdH covers this region. This completely blocks the cysteine-containing cavity in DdH, which is thus not accessible to the solvent medium. The surfaces were calculated with GRASP29 using a PDB file with internal water molecules removed and a probe radius of 1.4 Å.
The monomeric cytoplasmic hydrogenases are likely to be ancestral to the heterodimeric periplasmic ones because of the unusual topology of the small subunit in the latter (Fig. 1c). Translocation to the periplasm required gene splitting and signal-peptide insertions in the ancestral monomeric cytoplasmic enzyme.
Cysteine versus water molecules In DdH, a putative free cysteine amino acid is found in an internal pocket close
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to the protein surface, where it establishes hydrogen bonds with several residues, thus connecting the C and N termini of the large and small subunits (Fig. 2). Binding of this small molecule probably became necessary for the stabilization of these regions after the putative gene-splitting event in DdH that generated the small and large subunit. Accordingly, it is absent from the monomeric CpI, in which the corresponding pocket is occupied by four water molecules (Fig. 2b). Because there
The determination of the structure of the active site of Fe-only hydrogenases has revealed a new biological FeS cluster (Fig. 4). One of the unusual features of the active-site Fe binuclear center of both CpI and DdH structures is the presence of two terminal nonprotein ligands at each Fe atom. In the crystallographic analyses, it was assumed that these ligands represented CN2 or CO, or both, because of similarities between the Fourier-transform infrared (FTIR) spectra of Fe-only and NiFe hydrogenases9. [As mentioned above, studies involving X-ray diffraction and FTIR spectroscopy have established that the Fe site of NiFe hydrogenases contains one CO and two CN2 (Fig. 5c).] These observations have recently been confirmed by a study on the Fe-only hydrogenase from Desulfovibrio vulgaris, which indicated that this enzyme should contain at least one CO and one CN2 as terminal ligands to an Fe atom16. Because X-ray analysis cannot discriminate between CO and CN2, thanks to the small difference in the number of electrons of these two moieties, their assignment must rely on indirect evidence. One important aspect is the protein environment: the charged CN2 ligands are more likely to form hydrogen bonds than to sit on hydrophobic pockets; CO ligands can probably do both. In the active sites of CpI and DdH, hydrogen bonding at the diatomic moiety on the Fe atom distal to the [4Fe–4S] cluster (Fe2) is provided by the Ne of K358 and K237, respectively. Consequently, this diatomic ligand has been tentatively assigned as CN2 (Figs 4 and 5a,b). In the CpI structure, one diatomic moiety on the Fe atom next to the [4Fe–4S] cluster (Fe1) is hydrogen bonded to the Og of S232. Interestingly, this residue is not conserved in the DdH enzyme, in which the equivalent residue is A109. However, hydrogen bonding occurs at this position through the peptide-bond amide of the alanine.
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TIBS 25 – MARCH 2000 Differences in hydrogen bonding to the putative cyanide ligands might be important in dictating the midpoint potential of the binuclear Fe center and this is one of the factors that should determine the direction of the reaction (either hydrogen oxidation or hydrogen production). Another determining factor could be the local hydrogen concentration. The other two terminal diatomic ligands occupy hydrophobic pockets and have been modeled as CO. The presence of hydrogen-bond donors in close proximity to two of the diatomic ligands of the Fe site of the NiFe hydrogenase was the basis for the tentative assignment of CN2 rather than CO in that structure8. The active site Fe1 and Fe2 are bridged by two S atoms that are located 3.0 Å apart and are connected by a moiety of undetermined identity (Fig. 4). This moiety was originally identified in the CpI structure as a water molecule located ~2.7 Å from each S atom. However, the considerable residual electron density in difference electron density maps (using experimental against calculated diffraction data) suggests that the moiety consists either of a heavier atom at this position or of multiple atoms that covalently link the two S atoms. For the 1.6-Å-resolution structure of DdH, the electron-density map clearly indicates three covalently bound light atoms bridging the two sulfur atoms. The resulting small molecule has been modelled as a 1,3 propanedithiolate (PDT). It should be pointed out, however, that it is not possible to distinguish unambiguously between C, N and O, and so a combination of light atoms other than the three carbons proposed in PDT cannot be ruled out. This is especially true for the central atom, which makes close contact with the Sg of C178 in DdH. An equivalent three-atom linkage would be consistent with the data observed in the CpI structure, and it is probable that the linkages in both enzymes are the same. A covalent connection between the two S atoms is bound to have an impact on the electronic properties and the net charge of the H cluster. A feature that is reportedly different between the two structures is the identity of the active-site-Fe-bridging organic ligand. In CpI, the crystallographic refinement clearly indicates that it is a diatomic ligand, presumably CO, but the electron density in DdH is best modeled as an asymmetrically coordinated water molecule. However, the observed Fe-to‘water’ distance of 2.6 Å in DdH argues against an aqueous ligand and favors
Figure 4 Comparison of selected amino acid residues in the environment of the 2Fe subcluster of the H cluster of (a) Clostridium pasteurianum Fe-hydrogenase I (CpI) and (b) Desulfovibrio desulfuricans Fe hydrogenase (DdH). The [4Fe–4S] subcluster and associated Cys ligands are included to provide the proper perspective and are shown in gray. Fe atoms of the 2Fe subcluster, large red spheres; sulfur, yellow; oxygen, small red spheres; nitrogen, blue; carbon, white. A question mark indicates the unassigned moiety that bridges two sulfur atoms in CpI; a propane linkage has been putatively assigned at the analogous position in DdH. This figure was prepared with MOLSCRIPT26, MINIMAGE27 and RASTER3D28.
Figure 5 Comparison of the active site 2Fe subclusters of (a) Clostridium pasteurianum Fe-hydrogenase I (CpI), (b) Desulfovibrio desulfuricans Fe hydrogenase (DdH) and (c) the bimetallic active site of the Desulfovibrio gigas NiFe hydrogenase. The color scheme is the same as that shown in Fig. 4, except that the S bridging moiety in CpI is represented as a magenta sphere. This figure was prepared with MOLSCRIPT26, MINIMAGE27 and RASTER3D28.
a partially disordered, asymmetrically bound, diatomic ligand instead. FTIR studies of the D. vulgaris Fe-only hydrogenase suggest that a bridging CO is present in the oxidized state but absent when the enzyme is reduced16. Because the crystalline DdH is likely to be more reduced than the equivalent CpI (see below), the differences between the two active sites, as far as the bridging ligand is concerned, could simply reflect variations in their redox states. Thus, neither the X-ray analysis nor the FTIR studies can eliminate the possibility that the bridging ligand could alternate between an Fe-to-Fe bridging and an Fe terminally bound state during turnover. These questions, as well as the details of the chemical mechanism, will only be answered by additional biochemical and structural studies of the individual oxidation states.
Catalytic mechanism The details of the structure of the Fe binuclear center of the H cluster from both CpI and DdH suggest possible mechanisms for reversible hydrogen oxidation at the site. This center is remarkably similar in the two structures, but there are several subtle differences that are important to the mechanism. As suggested above, the main differences in the two structures are probably attributable to the oxidation state. Although it has not been within our means to characterize the oxidation state of the hydrogenase crystals directly, the CpI active site structure probably represents an anaerobically oxidized state and the D. desulfuricans structure a more-reduced state. This seems to be the most likely scenario for two reasons: first, crystals were grown under different conditions (CpI under
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REVIEWS conditions where the reductant present would be rapidly consumed and DdH under a 10% hydrogen, 90% nitrogen atmosphere, keeping a reducing environment) and second, there are no apparent open coordination sites in CpI for hydride formation (Figs 4, 5a,b). The coordination environment of the CpI H-cluster consists of two Fe atoms with relatively strong ligands (CO, CN2 and S) and a weaker ligand (H2O). The presence of the single weaker ligand on Fe2 makes it a very attractive site for displacement and formation of a bound hydride intermediate; indeed, recent results indicate that the competitive inhibitor CO binds to Fe2 (Ref. 17). In DdH, the site appears to be vacant, indicating either that it is unoccupied or that it corresponds to the site of hydride formation; a hydride would not be observable in the electron-density maps, even at 1.6 Å resolution. At any rate, all the available data are consistent with the involvement of this site in catalysis.
Substrate access to the active site There is a continuous hydrophobic channel running from the molecule’s surface to the active site, where it points at the apparently vacant Fe2 coordination site in the DdH structure10. This channel is also present in the CpI enzyme. Furthermore, the amino acids that line the channel are strictly conserved in the two Fe hydrogenases, suggesting that the same internal pathway is used for either the uptake or the production of molecular hydrogen. The channel is analogous to those observed in the Desulfovibrio fructosovorans NiFe hydrogenase. These channels have been shown to be likely to facilitate hydrogen access to the active site in this class of enzyme18.
Proton transfer The reaction intermediates of the production or uptake of hydrogen by hydrogenases are protons and hydrides3–6. These species result either from the transport of protons and their reduction by the buried active site (production) or from the heterolytic cleavage of the hydrogen molecule (uptake). In both instances, it is necessary to postulate the existence of proton-transfer pathways either to or from the solvent medium because the active sites are buried in the protein core. The CpI and DdH structures reveal several potential proton donor and acceptor groups near the active site of the two Fe hydrogenases10,11. The most obvious possibilities are conserved in both structures and
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TIBS 25 – MARCH 2000 include the following: (1) A lysine residue (K358 in CpI, K237 in DdH) that, as indicated above, forms a hydrogen bond with one of the putative CN2 ligands and is at ~4 Å from Fe2 (the putative hydrogen-binding/protonreduction site, see above). (2) A cysteine thiol (C299 in CpI, C178 in DdH) is ~5.0 Å from Fe2 and, in CpI, is within hydrogen-bond distance of the terminally bound water molecule coordinated at this site. (3) The bridging thiolates of the 2Fe cluster, for which protonation is not energetically favorable19 and might disrupt the binuclear Fe center, so their role as proton donors or acceptors is unlikely. The lysine residue mentioned above is the closest potential donor–acceptor molecule to Fe2. If K237 is indeed the catalytic base that assists in the heterolytic cleavage of molecular hydrogen, then a plausible proton-transfer pathway exists in DdH involving E240, three water molecules and E245 at the molecule’s surface. The involvement of this lysine residue as a general acid–base would modify the hydrogenbonding structure of the putative CN2 ligand of Fe2. It might be possible to detect such modification using FTIR for well-defined redox states. In CpI, a water molecule is close (~3.5 Å) to a free cysteine residue (C299) that lies ~5.0 Å from Fe2. It seems reasonable that hydride formation could occur by the displacement of the bound water molecule at this site, with the cysteine-bound water molecule acting as a proton donor– acceptor. The loss of the coordination of the water molecule by the distal Fe atom is consistent with the DdH structure, which reveals an apparent open coordination site at this position. A potential problem is the van der Waals interaction between the S of this cysteine and the central atom of the putative PDT (d 5 3.2 Å; d, distance). However, as mentioned above, if the latter atom is not C but either O or N, the observed distance would not be a problem. Thus, the cysteine residue is an attractive prospect as a proton donor–acceptor with a pKa of ~8.0 and strict conservation among all Fe-only hydrogenases for which the primary sequence has been determined12,13,20–25. Additional experimental investigation will be required to assign the proton donor–acceptor(s) definitively in the Fe-only hydrogenases.
Conclusions In spite of the fact that Fe-only hydrogenases from fermenting and
sulfate-reducing bacteria catalyse opposed reactions, they display many common features: the F- and H-domains have very similar foldings, and the active sites are almost identical. Major differences seem to derive from the localization of the enzymes. In DdH, a genesplitting event has resulted in the addition of one, or perhaps two, signal sequences that are needed for translocation to the periplasm. CpI has two extra domains at its N-terminal region that coordinate FeS clusters and may be instrumental in the interactions of the hydrogenase with redox partners. Residues surrounding the active site suggest ways through which protons can arrive at or departure from the active site. One of the Fe atoms of the binuclear center (Fe2) seems to be an excellent candidate for the initial substrate-binding site. A comparison between the active sites of NiFe and Fe-only hydrogenases (Fig. 5) strongly suggests that a low-spin Fe(II) with CO and CN2 coordination is central to hydrogen metabolism, perhaps as a hydride-binding species.
References 1 Stephenson, M. and Stickland, L.H. (1931) Hydrogenase: a bacterial enzyme activating molecular hydrogen. I. The properties of the enzyme. Biochem. J. 25, 205–214 2 Berger, D.J. (1999) Fuel cells and precious-metal catalysis. Science 286, 49 3 Schlegel, H.G. and Schneider K. (1978) In Hydrogenases (Schlegel, H.G. and Schneider, K., eds), pp. 15–44, Erich Goltze KG, Göttingen, Germany 4 Adams, M.W. et al. (1980) Hydrogenase. Biochim. Biophys. Acta 594, 105–176 5 Adams, M.W. (1990) The structure and mechanism of iron-hydrogenases. Biochim. Biophys. Acta 1020, 115–145 6 Albracht, S.P. (1994) Nickel hydrogenases: in search of the active site. Biochim. Biophys. Acta 1188, 167–204 7 Volbeda, A. et al. (1995) Crystal structure of the nickeliron hydrogenase from Desulfovibrio gigas. Nature 373, 580–587 8 Volbeda, A. et al. (1996) Structure of the [NiFe] hydrogenase active site: evidence for biologically uncommon Fe ligands. J. Am. Chem. Soc. 118, 12989–12996 9 Happe, R.P. et al. (1997) Biological activation of hydrogen. Nature 385, 126 10 Nicolet, Y. et al. (1999) Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Struct. Fold. Des. 7, 13–23 11 Peters, J.W. et al. (1998) X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853–1858 (Errata: 283, 35; 283, 2102) 12 Meyer, J. and Gagnon, J. (1991) Primary structure of hydrogenase I from Clostridium pasteurianum. Biochemistry 30, 9697–9704 13 Hatchikian, E.C. et al. (1999) Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757. J. Bacteriol. 181, 2947–2952 14 Pereira, I.A.C. et al. (1998) Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfuvibrio sp. J. Biol. Inorg. Chem. 3, 494–498 15 Nivière, V. et al. (1992) Site-directed mutagenesis of the hydrogenase signal-peptide consensus box prevents export of a b-lactamase fusion protein. J. Gen. Microbiol. 138, 2173–2183 16 Pierik, A.J. et al. (1998) A low-spin iron with CN and CO as intrinsic ligands forms the core of the active site in [Fe]-hydrogenases. Eur. J. Biochem. 258, 572–578
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TIBS 25 – MARCH 2000 17 Lemon, B.J. and Peters, J.W. (1999) Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum. Biochemistry 38, 12969–12973 18 Montet, Y. et al. (1997) Gas access to the active site of Ni–Fe hydrogenases probed by X-ray crystallography and molecular dynamics. Nat. Struct. Biol. 4, 523–526 19 Niu, S. et al. (1999) Theoretical characterization of the reaction intermediates in a model of the nickel–iron hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. 121, 4000–4007 20 Gorwa, M.F. et al. (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J. Bacteriol. 178, 2668–2675
21 Malki, S. et al. (1995) Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans. J. Bacteriol. 177, 2628–2636 22 Stokkermans, J. et al. (1989) hyd gamma, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase. FEMS Microbiol. Lett. 49, 217–222 23 Voordouw, G. et al. (1985) Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH2-terminal sequence. Eur. J. Biochem. 148. 509–514 24 Voordouw, G. et al. (1989) Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello. J. Bacteriol. 171, 3881–3889
Eukaryotic DNA polymerases, a growing family Ulrich Hübscher, Heinz-Peter Nasheuer and Juhani E. Syväoja In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called a, b, g, d, e, z, h, u and i), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase. ANY LIVING CELL and organism is faced with the tremendous task of keeping the genome intact in order to develop in an organized manner, function in a complex environment, divide at the right time and die when it is appropriate. To achieve this, DNA synthesis is required to duplicate the genetic information prior to cell division. DNA synthesis is also needed during DNA repair processes, including DNA recombination and bypassing lesions when the DNA has been damaged (translesion DNA synthesis). DNA synthesis is performed by enzymes, called DNA polymerases (DNA pol)1. Since the U. Hübscher is at the Institute of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; H-P. Nasheuer is at the Institute of Molecular Biotechnology, Dept of Biochemistry, Beutenbergstrasse 11, D-07745 Jena, Germany; and J.E. Syväoja is at the Biocenter Oulu and Dept of Biochemistry, University of Oulu, FIN-90570 Oulu, Finland, and Dept of Biology, University of Joensuu, FIN-80100 Joensuu, Finland. Email: [email protected]
discovery of DNA pol a in eukaryotic cells in 1957, the number of DNA pols identified has grown. In the early 1970s, DNA pol b and g were discovered leading to the simple concept that DNA pol a is the enzyme involved in DNA replication, DNA pol b in DNA repair and DNA pol g in mitochondrial DNA replication. However, the discovery of DNA pol d and e in mammalian cells during the 1980s complicated this interpretation. It also suggested that a particular DNA pol might have more than one functional task in a cell, and that a particular DNA synthetic event can require more than one DNA pol (reviewed in Ref. 2). Genetic studies performed with budding yeast Saccharomyces cerevisiae, for example, showed that the three DNA pols a, d and e share the task of replicating the cellular genome, and that DNA repair events such as base excision repair might require not only DNA pol b but also DNA pol d or DNA pol e, or both, especially in longpatch base excision repair. Because both replication and repair are of primary importance for cells and organisms, it
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
25 Santangelo, J.D. et al. (1995) Characterization and expression of the hydrogenase-encoding gene from Clostridium acetobutylicum P262. Microbiology 141, 171–180 26 Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 27 Arnez, J.G. (1994) MINIMAGE: a program for plotting electron-density maps. J. Appl. Crystallogr. 27, 649–653 28 Merritt, E.A. and Bacon, D.J. (1997) Raster3D photorealistic molecular graphics. Methods Enzymol. 277, 505–524 29 Nicholls, A. (1992) GRASP: Graphical Representation and Analysis of Surface Properties, Colombia University
appears that nature created safety mechanisms by employing various DNA pols for similar functional tasks. Translesion DNA synthesis, for example, requires at least DNA pols z and h, the former probably being responsible for error-prone translesion DNA synthesis and the latter performing error-free DNA translesion synthesis (reviewed in Ref. 3). In many cases, DNA pols have complex polypeptide structures because, in addition to the polymerizing subunit (that often contains a proofreading 39→59 exonuclease), they comprise other functional subunits (Table 1). These functions include other enzymatic activities (e.g. DNA primase) or allow the DNA polymerase to interact with other proteins, involved in check-point function, cell cycle control, DNA replication or DNA repair. The replicative DNA pols d and e, for example, are chaperoned by the accessory proteins replication factor C (RF-C), and proliferating cell nuclear antigen (PCNA). The interaction of these two accessory proteins with the DNA pols permits a high speed of DNA synthesis coupled with a high accuracy4. At the structural level, DNA pols appear to possess a universal DNA polymerase active site5. This is achieved by a two-metal-ion-catalysed mechanism and guarantees the incorporation of the correctly base-paired deoxyribonucleoside triphosphate (according to the Watson– Crick base pairing rule A–T and G–C) onto a growing template. However, DNA pols do differ in various aspects of their structural architecture5, as a result of the many possible interactions of DNA pols with other proteins and enzymes. Thus, the active site of the DNA pol is very conserved in evolution, whereas the structure of the surface of the molecules might differ considerably.
Functional roles of DNA pol a
DNA polymerase a–primase (DNA pol a– prim) has an important role in DNA replication (Fig. 1, reviewed in Ref. 6).
PII: S0968-0004(99)01523-6
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