Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation1

doi:10.1006/jmbi.2000.4136 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 303, 329±344

Comparison of Three Methyl-coenzyme M Reductases from Phylogenetically Distant Organisms: Unusual Amino Acid Modification, Conservation and Adaptation Wolfgang Grabarse1,2, Felix Mahlert2, Seigo Shima2, Rudolf K. Thauer2 and Ulrich Ermler1* 1

Max-Planck-Institut fuÈr Biophysik, Heinrich-HoffmannStraûe 7, 60528 Frankfurt, Germany 2

Max-Planck-Institut fuÈr terrestrische Mikrobiologie Karl-von-Frisch-Straûe 35043 Marburg, Germany

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisul®de of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37  C) and Methanopyrus kandleri (growth temperature optimum, 98  C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65  C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenÊ resolution of the zyme M and coenzyme B. The electron density at 1.6 A M. barkeri enzyme revealed that four of the ®ve modi®ed amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modi®cations may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was re¯ected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations. # 2000 Academic Press

*Corresponding author

Keywords: methyl-coenzyme M reductase; amino acid methylation; thiopeptide; methanogenesis; hyperthermophilicity

Introduction Biological methane formation is a process taking place in most anaerobic freshwater biotopes. It is carried out by methanogenic archaea which generate methane by reduction of CO2 with H2 and by disproportionation of acetate to CO2 and methane (Wolfe, 1996; Thauer, 1998). Like carbon dioxide, methane is a greenhouse gas contributing to the global warming effect. Signi®cant amounts of methane are produced in rice®elds, wastegrounds

and other biotopes created by humans, accounting for a considerable part of the anthropogenic greenhouse gas emissions (Minami & Takata, 1997; Millich, 1999). Methyl-coenzyme M reductase (MCR) is the terminal enzyme of the pathway of biological methane formation in all methanogenic archaea. It catalyzes the formation of methane and the heterodisul®de of coenzyme M and coenzyme B from methyl-coenzyme M and coenzyme B (Ellermann et al., 1988; Bobik et al., 1987): CH3 -S-CoM ‡ HS-CoB ! CH4 ‡ CoM-SS-CoB

Abbreviations used: MCR, methyl-coenzyme reductase; F430, factor 430, the nickel porphinoid of MCR. E-mail address of the corresponding author: [email protected] 0022-2836/00/020329±16 $35.00/0


G0 ˆ ÿ45 kJ=mol The enzyme has been isolated from Methanosarcina barkeri (strain Fusaro) (this paper), Methanobacter# 2000 Academic Press

330 ium thermoautotrophicum strains Marburg (Ellermann et al., 1989) and
H (Ellefson & Wolfe, 1981), Methanosarcina thermophila (Jablonski & Ferry, 1991) and Methanopyrus kandleri (Rospert et al., 1991). Two isoforms of the enzyme exist in the Methanobacteriales and Methanococcales (Thauer, 1998) which were shown to differ in their apparent KM and Vmax values (Bonacker et al., 1993). The gene expression level of the isoenzymes is differentially regulated by the growth conditions (Bonacker et al., 1992; Pihl et al., 1994; Pennings et al., 1997). The amino acid sequence of MCR is highly conserved among all methanogenic archaea, although some of the organisms are phylogenetically only very distantly related (Reeve et al., 1997; NoÈlling et al., 1996; Springer et al., 1995).

MCR from Three Methanogenic Archaea

Methyl-coenzyme M reductase is an enzyme of a molecular mass of 300 kDa with an (abg)2 subunit structure (see Figure 1(a)). The enzyme molecule contains non-covalently bound two mol of the nickel porphinoid factor 430 (F430). The nickel in F430 can exist in different oxidation states (Telser et al., 2000), (see Thauer, 1998 for a review). In methyl-coenzyme M reductase, the enzyme activity was shown to be proportional to the presence of the nickel in the Ni(I) oxidation state (Goubeaud et al., 1997). The crystal structure of MCR from M. thermoÊ autotrophicum has been determined at 1.45 A resolution (Ermler et al., 1997a). The enzyme was shown to be built up from two structurally identical active sites, each comprising residues of four

Figure 1. (a) Fold and subunit structure of the methyl-coenzyme M reductase. The a subunits are shown in blue and turquoise, the b subunits in red and orange and the g subunits in green and light green. The cofactor F430 is shown with the magenta space-®lling model. (b) Comparison of the Ca trace of methyl-coenzyme M reductase from three methanogenic archaea. MCR from M. barkeri (blue) possesses two additional loops (red arrows) which are not present in the enzymes from M. thermoautotrophicum (green) and M. kandleri (red). The Figure was prepared using the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merrit & Murphy, 1994).

331

MCR from Three Methanogenic Archaea

subunits a, a0 , b and g, and a molecule of the cofactor F430 (see Figure 1(a)). The cofactor is accessible Ê long from the bulk solvent only through a 50 A channel that is completely locked after the binding of the second substrate coenzyme B. Two inactive enzyme states, designated MCR-ox1-silent and MCR-silent were structurally characterized. In the MCR-ox1-silent state, coenzyme M and coenzyme B are found in the active site with the sulfhydryl group of coenzyme M being an axial ligand of the F430 nickel. In the MCR-silent state, the heterodisul®de CoM-SS-CoB coordinates the nickel of F430 with one of its sulfonate oxygen atoms. The electron density in all crystal structures of MCR from M. thermoautotrophicum clearly revealed the presence of four methylated amino acid residues, namely, 1-methylhistidine, 2-methylglutamine, 5-methylarginine and S-methylcysteine and of a thiopeptide bond in the active-site region of MCR (Ermler et al., 1997a). The nature of the modi®cations was con®rmed by MALDI-TOF spectroscopy and the methyl groups of the methylated amino acid residues were shown to be derived from methionine. Thus, the additional methyl groups are likely to be introduced into the protein by S-adenosylmethionine-dependent protein methylases rather than by an autocatalytical selfmethylation mechanism (Selmer et al., 2000). In this paper, we report on the crystal structures of methyl-coenzyme M reductase from M. barkeri and M. kandleri and compare them with the structure of MCR from M. thermoautotrophicum. We describe in detail the amino acid modi®cations in MCR from M. barkeri and M. thermoautotrophicum and draw conclusions regarding their possible relevance for the active-site geometry and the catalytic mechanism. For the comparative study these three organisms were chosen, since M. barkeri, M. thermoautotrophicum and M. kandleri are phylogenetically only very distantly related and possess different growth temperature optima (37  C, 65  C and 98  C) and intracellular salt concentrations (Shima et al., 1998). Structural features which are identical in the three enzymes are therefore of functional relevance, whereas observed structural differences may re¯ect both the phylogenetic drift and the adaptation of the proteins to their environment.

Results Purification, crystallization and structure determination of MCR from M. barkeri and M. kandleri MCR from M. barkeri was puri®ed anaerobically from methanol-grown cells by fractionated ammonium sulfate precipitation and a series of four chromatographic steps. The puri®ed enzyme was catalytically inactive, EPR silent and exhibited an UV/VIS spectrum typical for MCR with F430 in the Ni(II) oxidation state. N-terminal sequencing revealed the puri®ed enzyme to be the product of

the M. barkeri mcrBDCGA operon which had previously been sequenced (Bokranz & Klein, 1987). Crystals of MCR from M. barkeri were grown using a reservoir condition with PEG 5000 monomethylether as precipitant and glycerol as cryoprotectant. Crystals of the space group P21 and P212121 were obtained under similar crystallization conditions (see Methods for details). MCR from M. kandleri was pur®ed from H2/ CO2-grown cells by fractionated ammonium sulfate precipitation and by a series of three chromatographic steps. The N-terminal amino acid sequence (Rospert et al., 1991) matched that predicted from the mcrBDCDA operon in M. kandleri (NoÈlling et al., 1996). Crystals of the inactive enzyme were obtained with PEG 550 monomethylether as precipitant (see Methods for details). The crystals of MCR from M. barkeri were of the space group P212121. Diffraction data were colÊ at the BW7B beamlected to a resolution of 1.6 A line of the EMBL Hamburg (see Table 1 for details). The crystals of MCR from M. kandleri usually Ê resolution. Diffraction data diffracted only to 3.5 A Ê to 2.7 A resolution were collected from one crystal of MCR from M. kandleri with a completeness of 63 %. Due to the low completeness of the data, re®nement of the structure was only possible constraining the 2-fold non-crystallographic symmetry of the methyl-coenzyme M reductase molecule. The resulting model is less reliable than those of M. barkeri and M. thermoautotrophicum and was not interpreted in atomic detail apart from the activesite ligandation. Active-site ligands and several amino acid side-chains were veri®ed by simulated annealing omit maps.

Table 1. Data sets and re®nement statistics of MCR from M. barkeri and M. kandleri Data set Space group Unit cell Ê) a (A Ê) b (A Ê) c (A Ê) Wavelength (A Ê) Resolution (A Ê) Highest resolution shell (A Independent reflections Redundancya Completenessa (%) I/s (I)a Rmerge (%)c Radiation source

M. kandleri MCR

M. barkeri MCR

P212121

P212121

80.5 115.7 268.5 1.54 2.7 2.73-2.7 45,364 2.6 (2.2) 64.9 (62.2) 11.0 (3.8) 7.2 (18.6) RAXIS RU-200

113.7 153.1 153.3 0.84 1.6 1.62-1.6 326,507 2.7 (2.7) 93.5 (92.3) 16.2 (5.3) 5.9 (25.9) BW7Bb

a Values in brackets correspond to the highest resolution shell. b Beamline of the EMBL at DESY Hamburg. c Rmerge ˆ hklN sum over all j ˆ 1jIhkl ÿ Ihkl(j)j/hklN  Ihkl, re¯ections hkl and all observations N, with Ihkl(j) intensity of the j-th observation of re¯ection hkl and Ihkl mean intensity of the re¯ection hkl.

332

MCR from Three Methanogenic Archaea

The crystal structures of MCR from M. barkeri and M. kandleri were solved by the molecular replacement method with the programs AmoRe (Navaza, 1994) and CNS (BruÈnger et al., 1998) using the atomic model of MCR from M. thermoautotrophicum and the known amino acid sequences (Bokranz & Klein, 1987; NoÈlling et al., 1996). After rigid-body re®nement and simulated annealing with the program CNS an electron density was calculated which was of suf®cient quality to build the remaining parts of the models. Iterative model building and re®nement (see Methods for details) resulted in models with Rcryst/Rfree of 16.0 %/ 17.9 % in the case of MCR from M. barkeri and of 23.9 %/27.8 % in the case of MCR from M. kandleri (Table 2). Comparison of the methyl-coenzyme M reductase subunit folds As expected from the high level of sequence similarity (61-69 % identity), the three-dimensional structure of the three enzymes was found to be highly similar. The common fold of all methylcoenzyme M reductases and the superposition of the Ca traces of the enzymes from the three methanogenic archaea are shown in Figure 1(a) and (b). Ê The rms deviation of the Ca trace was 0.83 A between MCR from M. thermoautotrophicum and Ê between MCR from M. therM. barkeri and 0.74 A moautotrophicum and M. kandleri. In contrast, the rms differences of the Ca positions were only Ê in the active-site region of all around 0.1-0.2 A three enzymes, indicating almost identical active sites. The only substantial difference was the presence of two additional loops in the a subunit of MCR from the mesophilic M. barkeri which were not present in the enzymes from the thermophilic M. thermoautotrophicum and M. kandleri (Figure 1(b), red arrows). The ®rst loop extending from residues a18 to a28 is stabilized by three intrasubunit hydrogen bonds from Sera23 N to Glna48 O, from Asna24 Nd2 to Ilea83 O and from Phea21 N to Asna24 Od1 and by a hydrophobic interaction

Table 2. Parameters of the crystallographic models of MCR from M. barkeri and M. kandleri Data set Ê) Resolution (A Rcryst (%) Rfree (%) Number of protein atoms Number of solvent molecules Number of cofactor atoms Ê) Rmsd bond lengths (A Rmsd bond angles (deg.) Dihedral angles (deg.) Improper angles (deg.) Ê 2) Mean B factor (A Protein Solvent

MCR from M. barkeri

MCR from M. kandleri

30-1.6 16.0 17.9 18,948 2197 180 0.005 0.9 25.5 3.06

30-2.7 23.9 27.8 19,228 180 0.005 0.8 24.8 1.55

13.7 23.4

26.3 -

between the aromatic ring of Phea21 and Proa85. The second loop is located between residues a371383 and consists of three consecutive b turns which are stabilized by two intrasubunit hydrogen bonds between Glya378 N and Glya518 O and between Lysa379 Ne and Arga519 O. Apart from these differences, the folding of the subunits appeared to be almost identical between the three proteins. The active sites of MCR from M. barkeri and M. kandleri MCR from M. barkeri contained coenzyme M and coenzyme B as active-site ligands in a position nearly identical to that observed in the MCR-ox1silent state of the enzyme from M. thermoautotrophicum (Figure 2(a)) (Ermler et al., 1997a). The nickel center of F430 is coordinated by the coenzyme M sulfhydryl group from one side and by the oxygen atom of a glutamine side-chain from the other. After re®nement of the structure, residual electron density accounting for less than 10 % of the active sites was discovered that corresponds to MCR in complex with the heterodisul®de CoM-SS-CoB in the same position as observed in the structure of MCR from M. thermoautotrophicum in the MCR-silent state (Figure 2, red model) (Ermler et al., 1997a). In this state CoM-SS-CoB coordinates the F430 nickel with its sulfonate group. To form this enzyme state, the coenzyme M moiety must be rotated by an angle of nearly 90  relative to the position observed in the enzyme state with the reduced coenzymes M and B. The hydrogen bonding pattern of the cofactor F430 and of the substrate-binding sites was completely conserved in all MCR structures. The active site of MCR from M. kandleri (Figure 2(b)) was also found to contain the reduced coenzymes M and B analogous to the MCR-ox1-silent state in M. thermoautotrophicum. The modified amino acid residues in MCR from M. barkeri Surprisingly, the electron density of MCR from M. thermoautotrophicum revealed the presence of ®ve modi®ed amino acid residues, four methylated amino acid residues and one thiopeptide in the a subunit (Ermler et al., 1997a). Four of the ®ve modi®ed amino acid residues found in the structure of MCR from M. thermoautotrophicum were also present at the equivalent positions in MCR from M. barkeri. These are the 1-N-methylhistidine a271, the 5-methylarginine a285, the S-methylcysteine a472 and the thiopeptide bond at residue Glya465. Only the 2-methylglutamine found in MCR from M. thermoautotrophicum was not present in MCR from M. barkeri. Like in the crystal structure of MCR from M. thermoautotrophicum, the electron densities at the centers of the modi®ed atoms (Figure 3) were consistent with the additional atoms being fully occupied. No additional modi®ed amino acid residues were visible in the electron density. Due to the lim-

333

MCR from Three Methanogenic Archaea

Figure 2. Active sites of MCR from Methanosarcina barkeri and Methanopyrus kandleri. (a) 2Fo ÿ Fc Electron density Ê resolution of the active site of MCR from M. barkeri. Residual electron density between the sulfur atoms map at 1.6 A of the coenzymes M and B was observed that can be explained by the presence of small amounts of CoM-SS-CoB (red model) in the same conformation as observed in the structure of MCR from M. thermoautotrophicum in the MCRÊ effective resolution of the active site of MCR from M. kandleri. silent state. (b) 2Fo ÿ Fc Electron density map at 3.2 A Coenzyme M is the axial nickel ligand. To obtain an undisturbed acive-site view, the electron density of residue Phea439 was clipped off. The Figure was prepared using the program O (Jones et al., 1991).

ited quality of the data, the presence of the amino acid modi®cations could not be con®rmed for the MCR from M. kandleri. The geometry of the modi®ed amino acid residues and of their environment, which are described in the section below, did not differ between the enzymes from M. thermoautotrophicum and M. barkeri. As depicted in Figure 4, all of the modi®ed amino acid residues in methylcoenzyme M reductase are located near the activesite region or the binding site of the substrates. The modi®ed amino acid residues are completely conserved among the known MCR amino acid sequences. The same holds true for the residues that are in contact with the additional methyl groups and also for most of the surrounding

residues. To explore the possible functions of the modi®cations in the protein, their chemical environment is described below for the M. barkeri enzyme. If not stated otherwise, the neighboring amino acid residues are identical in the methylcoenzyme M reductases from M. barkeri and M. thermoautotrophicum. The thiopeptide at Glya a465 The most unusual amino acid modi®cation observed in MCR is the occurrence of a thiopeptide bond at residue Glya465 which is referred to as thioglycine. The thiopeptide is situated near the Ê Ni-S distance), between the bindactive site (12 A

Figure 3. 2Fo ÿ Fc Electron density maps at the 1s level of the modi®ed amino acid side-chains in MCR from M. barkeri. (a) 1-N-methylhistidine a271. (b) 5-Methylarginine a285. (c) Thiopeptide Glya465. (d) S-methylcysteine a472. The electron density at the 4s level was displayed to enable the comparison of the total electron densities of the thioglycine and the coenzyme B sulfur. The Figure was prepared using the program O (Jones et al., 1991).

334

MCR from Three Methanogenic Archaea

a472 forms two hydrophobic interactions with the side-chains of Hisb382 and Leua468, the latter being in contact with the catalytically important Asna501 and to the sulfur atom of thioglycine a465 (Figure 5(a)). 1-N-Methylhistidine a 271 The residue 1-N-methylhistidine a271 (p-methylhistidine, 3-N-methylhistidine according to the new IUPAC nomenclature; Cornishbowden, 1984) is part of the coenzyme B binding site. Its nonmethylated nitrogen atom Ne2 donates a hydrogen bond onto the oxygen atom of the coenzyme B phosphate group (Figure 5(b)). The short distance Ê and the geometry indicate the presence of of 2.6 A a hydrogen bond with fully overlapping orbitals. Unlike the amino acid residues methylcysteine a472 and methylarginine a285, the methyl group is orientated towards the solvent and does not have any contact with other protein residues. Figure 4. The modi®ed amino acid residues in MCR from M. barkeri. All the modi®ed residues (color coded) are found near the active or substrate binding sites. The substrates and cofactors are shown in magenta and the positions of the modi®cations are indicated by yellow arrows. The position which is only methylated in M. thermoautotrophicum is indicated by a red arrow. The Figure was prepared using the program SETOR (Evans, 1993).

ing sites of the coenzyme B and coenzyme M but without having direct contact to either of them (Figure 5(a)). The distance between the coenzyme B sulfur Ê , and atom and the thioglycine sulfur atom is 6.1 A the distance to the sulfonate oxygen atom OS1 of Ê . Geometric analysis of the coenzyme M is 7.9 A bond lengths and bond angles revealed that the thioglycine is present in a thiopeptide bond with a Ê between carbon and sulfur bond length of 1.66 A Ê between carbon and and a bond length of 1.34 A nitrogen. Interestingly, the sulfur of thioglycine Ê to the amide oxya465 has a distance of only 3.1 A gen atom of Asna501, whereas the amide nitrogen atom is in hydrogen bond distance to the sulfur atom of coenzyme B (Figure 5(a)). The electron densities of the amide group in the methyl-coenzyme M reductases of M. thermoautotrophicum and M. barkeri support the modelled amide bond orientation. S-Methylcysteine a 472 The S-methylcysteine a472 is not located in the active site but within the ®rst turn of an a helix which follows the b turn containing the thioglycine a465 and the main-chain nitrogen atom contacting the sulfonate oxygen atom of coenzyme M (Figure 5(a)). The methyl group of methylcysteine

5-(S)-Methylarginine a 285 The 5-methylarginine a285 is situated near the coenzyme B-binding site of MCR. Like the neighboring residue, Arga284, which is involved in coenzyme B binding, the residue methylarginine a285 is part of the substrate channel wall, although it does not have direct contact to the substrate coenzyme B. Its nearest distance to coenzyme B is Ê between the coenzyme B atom Ca and the 6.0 A methylarginine guanidyl nitrogen NH1 (Figure 6). The methylarginine donates a hydrogen bond to a solvent molecule which is in turn hydrogen bonded to the carboxyl group of coenzyme B. The guanidyl group of the methylarginine a285 is held in position by an intersubunit salt bridge to Glub183 and by an additional hydrogen bond to the atom Oe Asna494. The atom of the methylarginine with the largest solvent-accessible surface is the methylated atom Cd, whereas the methyl group itself is not solvent accessible and forms hydrophobic interactions with the residues Trpa332, Meta337 and Leua281 (Figure 6). The non-methylated glutamine a 420 The glutamine side-chains of the glutamine a420 in MCR from M. barkeri and the 2-methylglutamine a400 in MCR from M. thermoautotrophicum are located near the cofactor F430 (Figure 4, red arrow) Ê between the with the closest distance being 4.3 A atoms Cd of the glutamine and C6D of F430. Furthermore, the additional methyl group of the 2-methylglutamine a400 in M. thermoautotrophicum Ê to the atom Cd1 of the resihas a distance of 4.6 A due Tyra333 which contacts the axial nickel ligand with its phenolate oxygen atom (Figure 7). As the additional methyl group at the Ca atom of the 2methylglutamine a400 in MCR from M. thermoautotrophicum was not present in MCR from M. barkeri, the structure of both enzymes in the environment

MCR from Three Methanogenic Archaea

335

Figure 5. Chemical environment of the modi®ed amino acid residues in methyl-coenzyme M reductase from M. barkeri. (a) The thioglycine a465 (arrow) is located between the binding sites of the coenzyme M sulfonate group and the coenzyme B sulfhydryl group, but without having direct contact to either of them. The amide group of residue Asna501 is in close interaction with the thiopeptide sulfur. The additional methyl group (arrow) of the S-methylcysteine a472 in¯uences the environment of the residues Asna501 and thioglycine a465 via residue Leua468 and creates an additional hydrophobic intersubunit interaction with Hisb382. (b) The p-methylhistidine a271 donates a short hydrogen bond onto the coenzyme B phosphate moiety, whereas the additional methyl group (arrow) is directed towards the solvent. The Figure was prepared using the program SETOR (Evans, 1993).

of this modi®cation was analyzed to explore the consequences of the lack of the amino acid modi®cation (Figure 7). The environment of the glutamine residue in MCR from M. barkeri is distinct from that of MCR from M. thermoautotrophicum in that residue a423 is a threonine instead of an alanine residue, which donates a hydrogen bond onto a water molecule not present in M. thermoautotrophicum. The water molecule in turn donates a

hydrogen bond onto the oxygen atom of the catalytically important Tyra346. The position relative to F430 of the homologous tyrosines a333 and a346 in M. thermoautotrophicum and M. barkeri MCR was not signi®cantly changed between the species, indicating that the modi®cation did not have a dramatic effect on the active-site geometry. Comparison of the isotropic temperature factors of the Ca atoms near the modi®ed glutamine residue did not reveal signi®cant differences in the temperature factor distribution of the residues surrounding the additional methyl group in MCR from M. thermoautotrophicum.

Comparison of the solvent accessible surface and of the subunit interfaces

Figure 6. Chemical environment of the methylated arginine in methyl-coenzyme M reductase from M. barkeri. The additional methyl group (arrow) of the 5methylarginine a285 is surrounded by hydrophobic residues (shown in green). A water molecule bridges between the substrate coenzyme B and the guanidyl group of the methylarginine which forms an intersubunit salt bridge with Glub183 and a hydrogen bond with Asna494. The Figure was prepared using the program SETOR (Evans, 1993).

The strategy of M. thermoautotrophicum, M. kandleri and some other methanogens such as Methanothermus fervidus to adapt to high temperatures is to maintain a higher intracellular concentration of potassium and of cyclic 2,3-diphosphoglycerate (Shima et al., 1998). Thus, adaptation to higher temperatures and to high concentrations of lyotropic salt is coupled in these organisms (cf. Table 3). M. barkeri, M. thermoautotrophicum and M. kandleri are phylogenetically only distantly related, as shown by the comparison of the genes coding for the 16 S RNA (Burggraf et al., 1991). Common structural features in less sequence conserved regions at the protein surface of the thermophilic methyl-coenzyme M reductases should therefore re¯ect their adaptation to high temperatures and intracellular salt concentrations but not their phylogenetic relatedness. This is especially true since the

336

MCR from Three Methanogenic Archaea

re¯ected in the amino acid composition of the protein which contrasts with that of M. barkeri: The frequency of glutamic acid is signi®cantly increased, whereas the frequency of aspartic acid is in the same range in all three proteins (Table 3). An increased Arg:Lys ratio, which was reported to be a feature of haloadaptation (Pieper et al., 1998; Jaenicke, 1991; Cendrin et al., 1993), was not observed despite the fact that the intracellular concentration of salts in M. kandleri is above 2 M (Kurr et al., 1991; Shima et al., 1998). In Table 3 the accessible surface areas of the protein surface and of the intersubunit interfaces are compared. Whereas both the solvent-accessible surface area and the area of the intersubunit interface per atom are similar in all the three proteins, the hydrophobic portion of the solvent accessible surface area was found to be signi®cantly decreased in the MCR from the hyperthermophilic M. kandleri, but also in the protein from the moderately thermophilic M. thermoautotrophicum. In contrast, the intersubunit interface area of the MCR from M. kandleri showed only a slightly higher fraction of non-polar residues in its intersubunit interfaces compared with the other two proteins. To explore the adaptation of the amino acid residues at the protein surface, the fraction of the accessible surface contributed by each amino acid side-chain type was compared for the protein surface of the three MCR proteins (data not shown). The analysis revealed that the decrease in the hydrophobic surface portion of the hyperthermophilic MCR was solely caused by the oxygen atoms of glutamate residues. The decrease in the hydrophobic surface fraction was mostly contributed by exchange of short chain amino acid residues such as alanine, serine and threonine. In contrast, the hydrophobic surface contribution of large residues such as tryptophan, phenylalanine and tyrosine did not signi®cantly change. Near the protein surface of MCR from M. kandleri, a high number of amino acid exchanges leading to new hydrophobic

Figure 7. Superposition of the structures of MCR from M. barkeri (red) and M. thermoautotrophicum (green). Near the site of the modi®cation in MCR from M. thermoautotrophicum a threonine residue (a423) is found in the M. barkeri MCR that donates a hydrogen bond onto a water molecule which is not present in MCR from M. thermoautotrophicum. The water molecule in MCR from M. barkeri and also the methylated amino acid in MCR from M. thermoautotrophicum (black arrow) may in¯uence the position of the active site tyrosine a346 that points onto the F430 nickel ligand. The Figure was prepared using the program SETOR (Evans, 1993).

surface of MCR is only to a minor portion associated with catalytic functions. In Figure 8(a)-(c) the electrostatic surface potentials, as calculated with the program GRASP (Nicholls et al., 1991), are compared for the methylcoenzyme M reductase from the three different methanogens. It can be seen that the methyl-coenzyme M reductases from M. thermoautotrophicum and M. kandleri are similar in their highly negative electrostatic surface potential. This similarity is

Table 3. Cellular environment, selected amino acid frequencies and surface properties of MCR from three different methanogenic archaea MCR from: Intracellular salt concentration (M) Growth temperature optimum ( C) Amino acid content (%) Glutamate Aspartate Lysine Arginine No. of atoms (protein ‡ cofactor) Ê 2) Solvent accessible surface (A Hydrophobic portion (%) Ê 2) Subunit interface (A Ê 2) Subunit interface/atom (A Hydrophobic portion (%)

M. barkeri

M. thermoautotrophicum

M. kandleri

0.3 37

0.7 65

>2.0 98

5.53 6.57 4.53 4.81

8.32 6.46 4.68 4.36

9.87 6.39 5.50 4.36

19,083 58,740 54.7 50,388 2.64 60.5

19,298 61,054 50.8 49,833 2.58 60.2

19,510 60,851 50.4 51,173 2.62 61.8 Ê and standard All calculations were done with the program NACCESS (Hubbard & Thornton, 1993) using a probe radius of 1.4 A van der Waals radii.

337

MCR from Three Methanogenic Archaea

Figure 8. Electrostatic surface potential of MCR from three methanogenic archaea. (a) M. barkeri (37  C, 0.3 M intracellular salt concentration); (b) M. thermoautotrophicum (65  C, 0.7 M intracellular salt concentration); (c) M. kandleri (98  C, >2 M intracellular salt concentration). The electrostatic potential was calculated in the range from ÿ25 kT to 25 kT with the program GRASP (Nicholls et al., 1991). To obtain comparable results, the salt concentration was assumed to be 2 M in all calculations. (d) The conservation of the negatively charged residues in MCR mapped onto the structure of MCR from M. kandleri: Aspartate residues and glutamate residues common between the three structurally analyzed enzymes are shown in orange. The glutamate residues only present in the thermophilic M. thermoautotrophicum and M. kandleri enzymes are shown in red.

interactions was observed for instance between residues Trpa46 and Leua0 548 or between residues Trpa162 and Alaa543. As the presence of the glutamate side-chains seemed to be most important for the adaptation to the intracellular conditions, it was analyzed which of the glutamate side-chains were present only in the thermophilic enzymes, and whether the additional glutamate residues could be found at structurally equivalent positions. Only residues were analyzed that showed an rms deviation of Ê . In Figure 8(d) the Ca positions of less than 3.8 A the conservation of the glutamate positions in the MCR from M. kandleri is shown. Of the 232 glutamate residues in MCR from M. kandleri, 92 were present in all three crystal structures. Of the remaining 140 glutamates, 76 were found at structurally equivalent positions in M. kandleri and M. thermoautotrophicum, but not in M. barkeri. Thus, more than 50 % of the not conserved glutamate residues were found at structurally equivalent positions in the thermophilic enzymes. Given the large phylogenetic distance between the three species, this ®nding re¯ects a high degree of speci®city for the location of the glutamate residues at the protein surface. In contrast, only ten structurally aligned glutamate side-chains were common to M. kandleri and M. barkeri but not to M. thermoautotrophicum. Another 34 glutamate residues of

M. kandleri did not have a structural equivalent in either of the structures of MCR from M. thermoautotrophicum and M. barkeri.

Discussion The modified amino acid residues and their possible influence on the catalytic activity All the amino acid modi®cations observed in methyl-coenzyme M reductase so far have in common that they are located in the highly conserved active-site region of the protein. The presence of four out of ®ve amino acid modi®cations in two phylogenetically very distant methanogens may thus indicate that the common modi®cations are required for the enzyme to be catalytically effective. Although the exact function of the amino acid modi®cations is not yet known, analysis of the interactions of the modi®ed residues might help to explain their conservation over the large phylogenetic distance. The most exciting amino acid modi®cation is the occurrence of a thiopeptide between the residues Glya465 (Figure 5(a)) and Tyra466. One of the functions of this residue might be to adjust the orientation of the asparagine residue a501, such that it points with its amide nitrogen atom, but not with the oxygen atom onto the coenzyme B sulfhy-

338 dryl group and provides hydrogen bond stereochemistry. Because of this hydrogen bond, the pKa of the coenzyme B sulfhydryl group is decreased, which may facilitate the cleavage of the proton during the catalytic reaction. One can, however, imagine that this objective might also have been achieved by a non-modi®ed amino acid residue. Another possible function of the thioglycine is to work as a redox active one-electron-relay with the sulfur atom being alternatingly present as thioketon and as thioketyl anion radical. The location of the residue between the two substrate-binding Ê away from the nickel is compatible sites and 12 A with such a function. Support for the presence of a second redox active group in MCR besides the F430 nickel comes from the observation that the inactive MCR state MCR-ox1 probably contains the F430 in the nickel (I) oxidation state (Telser et al., 2000) but can be reductively converted to the active enzyme state MCR-red1 with Ti(III) citrate (Goubeaud et al., 1997). Furthermore, the distance of the thiopeptide Ê to the position of the disul®de sulfur of only 6 A bridge of the heterodisul®de CoM-SS-CoB is consistent with a function as an electron acceptor from a putative heterodisul®de anion radical that has been proposed to be an intermediate of the catalytic reaction (Ermler et al., 1997a; Thauer, 1998). The additional methyl group of the S-methylcysteine (Figure 5(a)) interacts with the side-chain of a leucine residue, which in turn interacts with the thioglycine. The presence of these methylcysteine residues in the crystal structures of MCR from two very distantly related organisms may thus also re¯ect the importance of the thiopeptide bond, which is the sole potentially catalytic relevant residue in the vicinity of the S-methylcysteine. The function of the methylation in methylhistidine a271 (Figure 5(b)) is probably associated with the binding of the substrate coenzyme B. In the coenzyme B binding site, the methylated histidine donates a hydrogen bond onto the phosphate group of coenzyme B (Figure 5(b)) whereas the additional methyl group is directed towards the solvent. The binding of the coenzyme B phosphate group may be in¯uenced via an altered strength of the hydrogen bond or via the slightly increased pKa of the methyl-histidine imidazole ring (Hartman & Hartman, 1992) which maintains the protonation of the imidazole ring at higher pH conditions. The introduction of the methyl group into the 5methylarginine (Figure 6) lead to additonal hydrophobic interactions with neighboring hydrophobic residues that are predicted to lock the arginine in the conformation found in the crystal structures. This may be necessary to prevent the arginine residue from protruding into the substrate channel where the charged guanidyl group could unfavorably interact with the sulfonate groups of either methyl-coenzyme M or the heterodisul®de CoMSS-CoB. It is, however, also possible that the conformation of the arginine is important for the

MCR from Three Methanogenic Archaea

shape of the substrate channel wall or for the formation of the intersubunit salt bridge (Figure 6). The methylation of the a carbon of the residue Glua400 in M. thermoautotrophicum was found near the active site tyrosine a346 which donates a hydrogen bond onto the axial nickel ligand. A possible function of this residue might therefore be to restrict the conformational ¯exibility of this tyrosine residue. In the M. barkeri enzyme, which does not contain this methylation, the cavity ®lled by the additional methyl group is occupied by a water molecule which is hydrogen bonded to a threonine residue, one of the few amino acid residues exchanged in the near active site region of MCR. Despite these differences, both the active site structures are very similar. One possible explanation for the lack of the methylation in the M. barkeri MCR is that the backbone methylation is only required in thermophilic methanogens. However, no signi®cant change in the temperature factor distribution of the active site that could support this hypothesis was observed at 90 K. Such differences might, however, become apparent at higher temperatures, whereas the crystal structures were determined at only 90 K. The modi®ed residues in MCR were proposed above to be involved in very different functions such as ligand binding, restraint of amino acid motions and electron transfer. Moreover, in MCR, even different types of interaction for methylations with the surrounding protein were found. Thus, the methyl groups of the methylarginine and the Smethylcysteine act via hydrophobic interactions, whereas the methyl group of the 1-N-methylhistidine changes the binding properties of MCR to coenzyme B via the inductive (I‡) effect. Whereas amino acid modi®cations have also been found in the active sites of other proteins (Yoshikawa et al., 1998; Holmes et al., 1990), the occurrence of ®ve differently modi®ed residues in the active-site region of one enzyme is unique so far. Possible mechanisms of amino acid modification The four methylated amino acids in M. thermoautotrophicum have been shown to arise from S-adenosyl-methionine rather than from the C1 source carbon dioxide in M. thermoautotrophicum (Selmer et al., 2000), and are thus not side-products of the catalytic reaction. Hence, the methylations must have been introduced into the enzyme by the action of speci®c protein methylases, some of which have been found in the genome of M. thermoautotrophicum
H (Smith et al., 1997) but are not yet characterized at the protein level. From the analysis of the crystal structures of MCR it is evident that the modi®cation of the fully assembled hexamer is impossible because the positions of the modi®cations are inaccessible to a modifying enzyme. Moreover, some of the atoms proposed to be attacked by a modifying enzyme, for instance the Ca atom of the methylglutamine

MCR from Three Methanogenic Archaea

are even buried in the structure of the folded a subunit. Thus, a methylation at the level of the nascent or partially folded subunit as shown for protein methylation in yeast (Niewmierzycka & Clarke, 1999) is more likely for these residues. As the enzymatic methylations of the carbon, nitrogen and sulfur atoms of the protein are expected to proceed rather differently we have explored whether similar amino acid modi®cations have been found elsewhere in nature. Whereas the methylation of the cysteine residues has been observed in proteins (Roehm & Berg, 1998), the 1N-methylhistidine was so far only found in biologically active peptides like anserine (b-alanyl-L-1methylhistidine). For the N-methylation of arginine residues which in¯uence the substrate speci®city of the RNA binding protein A1 in yeast (Zobelthropp et al., 1998; Kim et al., 1998) a consensus sequence R/L-A/G-R-A/G-R/L for the methylating enzyme was determined using a peptide library (Kim et al., 1998). The amino acid sequence of the methylcoenzyme M reductases near the 5-methylarginine in methyl-coenzyme M reductase is similar to that consensus sequence in that it contains two further arginine residues near the modi®ed arginine. The 5-methylarginine and the 2-methylglutamine in M. thermoautotrophicum are different from all other amino acid methylations observed in nature so far, in that they are C-methylations. The introduction of these modi®cations into the protein will require a mechanism of the S-adenosylmethioninedependent modi®cation different from that of the arginine-N-methyltransferases. One possible mechanism involving ylides has been proposed (Selmer et al., 2000). The origin of the sulfur in the thioglycine a465 is unknown. The closest analogon observed in nature are thiopeptide antibiotics like promoinducin (Yun & Seto, 1995) or thiostreptone (Porse et al., 1998) that are produced by Streptomyces species. Also, Cterminal amino acid sulfurylation is known to occur during the biosynthesis of thiamine and molybdopterine (Taylor et al., 1998; Appleyard et al., 1998). Thus, the thioglycine at position a465 is the ®rst occurrence of a thiopeptide bond in the polypeptide chain of a protein. The mechanism of modi®cation of this amino acid can be expected to be different to those already known.

Structural conservation and adaptation to the cellular environment The degree of amino acid conservation found in the enzyme methyl-coenzyme M reductase is unusually high and similar to that of the 16 S RNA (Springer et al., 1995). Not surprisingly, no larger differences were observed in the active-site geometry and the overall fold. The highly conserved regions not only comprise the residues contacting the substrates and cofactors but also most of the unusual amino acid modi®cations and many of the intersubunit contacts.

339 In the three methanogenic archaea discussed here, the adaptation to higher temperatures is coupled to increasing intracellular concentrations of potassium ions and 2,3-diphosphoglycerate (see Table 3) which is proposed to act as a thermoprotectant (Shima et al., 1998). However, in contrast to other protein structures (Auerbach et al., 1997; Yip et al., 1995) no differences were observed in either the protein fold, the ®xation of the N and C termini, in the number of salt bridges and in the contact area between the subunits. Although these characteristics may still be important for thermostability, they are not determinants of the differences between these proteins. MCR from M. barkeri possesses two additional loops in the a subunit which are not present in MCR from the thermophilic M. kandleri and M. thermoautotrophicum. However, as both the additional loops are stabilized by hydrogen bonds to the surrounding protein, it is not clear whether their absence can be associated with thermoadaptation as found in other proteins (Spiller et al., 1999) and by genome comparison (Thompson & Eisenberg, 1999). As the interior of the three proteins is exceptionally similar in amino acid sequence and structure, it is likely that both the high salt and temperature adaptations are primarily based on differences at or near the protein surface. The analysis of the protein surface of MCR from the three organisms (Table 3) showed that with higher intracellular salt concentrations the fraction of glutamate residues at the protein surface was increased, whereas the aspartate frequency did not signi®cantly change. This indicates that not only the negative charge but also the aliphatic arm length of the glutamate is required for high salt adaptation. The positions of the glutamate side-chains in the three-dimensional structure were found to be much more conserved between the thermophilic, salt-adapted M. thermoautotrophicum and M. kandleri enzymes than would be expected from the phylogenetical relatedness of the organisms. Thus, the fact that the conserved glutamate residues are evenly distributed over the protein surface and protrude towards the bulk solvent without having contact to other residues may also be of importance for their function in high salt adaptation, which may be based on the capability of the ¯exible negative charges to prevent aggregation (Elcock & McCammon, 1998) or on increased water-binding capcity of the glutamate residues (Frolow et al., 1996). The observed increase of the surface charge in MCR from the hyperthermophilic archaea was accompanied by a decrease of the hydrophobic surface area. A more careful analysis of the protein surface showed that this decrease did not lead to fewer hydrophobic interactions because only small hydrophobic residues were exchanged against the glutamate residues and additional hydrophobic residues were introduced beneath the protein surface, particularly in the M. kandleri enzyme. This is in agreement with larger contributions of intramolecular hydrophobic interactions to the protein

340

MCR from Three Methanogenic Archaea

stability at high salt concentrations due to the salting-out effect (Zaccai & Eisenberg, 1990; Ermler et al., 1997b) and at high temperatures (Lo Leggio et al., 1999). The methyl-coenzyme M reductases described here differ in the temperature dependence of the catalytic activity. For instance, the enzyme from the thermophilic M. thermoautotrophicum displays very low activity at 37  C, the growth temperature optimum of the mesophilic M. barkeri. However, both the enzymes possess almost identical active sites. Thus, the observed changes in the temperature dependence of the catalytic activity should either be triggered by very subtle changes in the active-site region such as the additional methylation at the glutamine a400 in M. thermoautotrophicum or be effected by the different properties of the surface regions. Different surface properties might in¯uence catalysis when substrate binding and product release are accompanied by changes in ¯exibility and conformation A possible explanation for the low activity of the M. thermoautotrophicum enzyme at 37  C may therefore be decreased ¯exibility of the outer parts of substrate channel of the empty enzyme compared with that of M. barkeri, which could obstruct substrate binding or product release. There was, however, no indication of different temperature factor pro®les of the substrate channels with coenzyme B bound between the enzymes of M. thermoautotrophicum and M. barkeri determined at 90 K.

ammonium sulfate. The column was developed using a linear ammonium sulfate gradient of 270 ml with a ¯ow rate of 3 ml/min in the range of 1.4 M to 0.0 M ammonium sulfate. The fractions containing MCR eluted at a concentration of approximately 0.2 M ammonium sulfate. The pooled fractions were applied to a Macro Prep Ceramic Hydroxyapatite column (Biorad, volume 2.51 cm2  10 cm), which was equilibrated with 10 mM potassium phosphate buffer at pH 7.0. Elution was performed using a ¯ow rate of 3 ml/min and a step gradient of potassium phosphate with steps of 20 ml 0 M; 40 ml 0.05 M; 40 ml 0.075 M; 40 ml 0.1 M; 40 ml 0.15 M; 40 ml 0.2 M; 40 ml 0.25 M; and 40 ml 0.5 M potassium phosphate at pH 7.0. MCR eluted at a potassium phosphate concentration of 0.1 M. The pooled fractions were loaded on a ResourceQ column (Amersham Pharmacia Biotech, volume 6 ml) which had been equilibrated with 50 mM Mops/KOH, pH 7.0. The column was developed with a step gradient of sodium chloride using steps of 20 ml 0 M; 20 ml 0.2 M; 20 ml 0.4 M; 20 ml 0.6 M; 20 ml 1 M; 40 ml 2 M NaCl. Puri®ed MCR was eluted at a concentration of 0.4 M NaCl. No contaminants were visible in the SDS PAGE stained with Coomassie Brillant Blue R250. As the enzyme activity was rapidly lost during the puri®cation procedure, the enzyme was identi®ed by its bound cofactor F430 in the early stages of the puri®cation. Fractions were tested for the presence of F430 by denaturation of the protein with 4 M guanidinium chloride and precipitation of the protein by titration with trichloracetic acid until a pH of 2 was reached. The protein was removed by centrifugation and the supernatant was applied to a RP18 HPLC column (Merck, Darmstadt, Germany) and the eluted F430 was detected UV spectrometrically at a wavelength of a420 nm.

Methods

N-Terminal sequencing of MCR from Methanosarcina barkeri

Purification of MCR from Methanosarcina barkeri

Subunits of MCR from M. barkeri (50 mg pure enzyme) were separated by HPLC on a Supelcosil TM-LC-3DPcolumn (4.6 mm  25 cm; Supelco, Bad Homburg). Elution was performed with a gradient of acetonitril in 0.1 TFA/water applied over 32 minutes starting from 0 % acetonitrile to 84 % (w/v) acetonitrile. Eluting substances were detected with a diode array (Hewlett Packard, Bad Homburg) recorded at the wavelengths of 280 nm and 420 nm, respectively. The separated g subunit was then subjected to Edman degradation, and the ®rst 20 N-terminal amino acid residues were determined.

All puri®cation steps were carried out anaerobically under an N2/H2 (95 %/5 %) atmosphere. Methanosarcina barkeri strain Fusaro (DSM 804) was grown on methanol as described (Karrasch et al., 1989). Samples of 30 g cells of Methanosarcina barkeri were suspended in 30 ml 50 mM Mops/KOH, pH 7.0 and disrupted by sonication at 4  C. After dilution of the suspension in a ratio of 1:2 with 50 mM Mops/KOH, pH 7.0, cell debris was removed by centrifugation at 10,000 g for 30 minutes. The supernatant was then subjected to ultracentrifugation at 140,000 g. Membrane-free cell extracts (100 ml) were subjected to fractionated ammonium sulfate precipitation using fractionation steps of 60 % and 100 % saturation and a buffer of 50 mM Mops/KOH, pH 7.0. The pellet was then redissolved in 50 ml 50 mM Mops/KOH, pH 7.0, applied to a desalting column (Amersham Pharmacia Biotech) and loaded on a Q-Sepharose column (high perfomance, 3.58 cm2  10 cm) which was equilibrated with 50 mM Mops/KOH, pH 7.0. The protein was eluted using a NaCl step gradient at a ¯ow rate of 3 ml/min with six 40 ml steps of 0.1 M, ranging from 0 to 0.6 M NaCl and three 40 ml steps corresponding to 0.8 M, 1 M and 2 M NaCl. The fractions containing MCR eluted at a 0.4 M NaCl concentration. The ammonium sulfate concentration of the samples was adjusted to 1.4 M and the fractions were loaded on a Phenyl Sepharose column (Amersham Pharmacia Biotech, volume 3.58 cm2  10 cm) which was equilibrated with 1.4 M

Crystallization of MCR from Methanosarcina barkeri Methyl-coenzyme M reductase prepared from M. barkeri cells grown on methanol was crystallized using the hanging drop method performed in a 24 well Linbro plate. The optimized reservoir solution contained 10 % (w/v) PEG 5000 monomethylether, 10 % (w/v) 2-propanol, 0.2 M ammonium acetate, 0.1 M magnesium acetate, 20 % (w/v) glycerol and 0.1 M 2-(N-morpholino)-ethanesulfonic acid (Mes)/KOH (pH 6.5). Droplets of 1 ml size of a solution containing 30 mg/ml of MCR from M. barkeri in 10 mM Tris-HCl (pH 7.0) and of the reservoir solution were thoroughly mixed. The temperature of the crystallization was 14  C. Flat, rodshaped crystals belonging to the space group P21 with Ê ; b ˆ 109.9 A Ê ; c ˆ 124.2 A Ê and cell constants of a ˆ 96.3 A b ˆ 102.9  appeared after several days. After four weeks

MCR from Three Methanogenic Archaea a second orthorhombic crystal form was obtained belonging to the space group P212121 with cell constants Ê ; b ˆ 153.1 A Ê and c ˆ 153.3 A Ê . The crystals of a ˆ 113.7 A of this form diffracted to atomic resolution and were used for structure analysis. Purification of MCR from Methanopyrus kandleri Cells of M. kandleri (AV19) (Rospert et al., 1991; Kurr et al., 1991) were a gracious gift from Prof. K. O. Stetter (University of Regensburg, Germany). Frozen cells (approximately 20 g wet mass) of M. kandleri were suspended in 50 ml 50 mM Mops/KOH, pH 7.0 containing 10 mM MgCl2 and 20 mg DNAseI and subsequently passed three times through a French pressure cell at 110 MPa under strictly anaerobic conditions. Cell debris was removed by anaerobic centrifugation at 29,000 g for 30 minutes. The supernatant was then subjected to ultracentrifugation for ten minutes at 140,000 g. After ®ltration with a 0.45 mm ®lter membrane (Millipore, Eschborn, Germany), the ®ltrate was anaerobically applied to a 26/10 Phenyl Sepharose High Performance column (Amersham Pharmacia Biotech) which was equilibrated with 50 mM Mops/KOH (pH 7.0) containing 1.3 M ammonium sulfate (¯ow rate 2 ml/min). The ¯owthrough containing the methyl-coenzyme M reductase was collected, dialyzed against 50 mM Mops/ KOH, pH 7.0 and concentrated by ultra®ltration with a PM30 membrane (Millipore, Eschborn, Germany) and then aerobically applied to a Mono Q HR10/10 column (Amersham Pharmacia Biotech) which was previously equilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.3 M NaCl with a ¯ow rate of 3 ml/min at a temperature of 4  C. The column was washed with 20 ml equlibration buffer and eluted with a linear gradient of NaCl (0.4 M-0.68 M, 72 ml). Methyl-coenzyme M reductase typically eluted at NaCl concentration of 0.5-0.58 mM. The fractions containing the enzyme were pooled, concentrated by ultra®ltration with a PM 30 membrane and dialyzed against 10 mM potassium phosphate (pH 7.0). The enzyme was then applied to a 1.3 cm2  10 cm Macro Prep Ceramic Hydroxyapatite column (Biorad Laboratories, Munich, Germany) which was equilibrated with 10 mM potassium phosphate, pH 7.0 at a ¯ow rate of 3 ml/min at 4  C. MCR was eluted using a linear gradient of potassium phosphate (0.01 M-0.5 M, 260 ml). The fractions containing the enzyme (0.17 to 0.21 mM potassium phosphate) were concentrated to 50 mg/ml and dialyzed against 10 mM Tris-HCl (pH 7.8). Crystallization of MCR from Methanopyrus kandleri Methyl-coenzyme M reductase from M. kandleri was crystallized using the hanging drop method and a reservoir solution containing 10 % PEG 550 monomethylether, 100 mM NaCl, 20 mM MgCl2 and 100 mM Hepes (pH 7.0). Drops of 3 ml of the puri®ed protein (20 mg/ml in 10 mM Tris, pH 7.8) and of the reservoir solution were mixed and stored at a temperature of 13  C. The rod shaped crystals of the space group P212121 with cell Ê ; b ˆ 115.7 A Ê ; cˆ 268.5 A Ê typically constants of a ˆ 80.5 A appeared after two weeks. Whereas large crystals were generally not suitable for X-ray crystallography due to their high mosaicity, some of the small crystals exhibited lower mosaicities and improved diffraction behavior. The crystals of MCR split when transferred into a solution containing either glycerol or ethyleneglycol as cryoprotectants. Thus, the measurements had to be

341 carried out with small fragments obtained from larger crystals. Data collection, model building and refinement of the crystal structure of MCR from Methanosarcina barkeri The dataset of MCR from M. barkeri was collected at the BW7B beamline of the EMBL at DESY Hamburg using a MAR345 image plate detector (MAR Research, Hamburg) at a temperature of 100 K supplied by a cryojet cooler (Oxford Cryosystems, Oxford, UK). All diffraction data were processed with the programs of the HKL(Otwinowski & Minor, 1997) and CCP4-suites (CCP4, 1994). The crystal structure of MCR from M. barkeri was solved by the molecular replacement method with the program AMoRe (Navaza, 1994). Using the complete model of MCR from M. thermoautotrophicum and the Ê resolution, a unique solution data between 10 and 4 A with an R-factor of 43 % was obtained. The coordinates of MCR from M. thermoautotrophicum including the solvent molecules but not the cofactors coenzyme M and coenzyme B were transformed according to the molecular replacement solution. Prior to re®nement, 5 % of the re¯ections were separated for crossvalidation (BruÈnger, 1992). No non-crystallographic symmetry restraints were applied during the re®nement procedure. After rigidbody re®nement with the program CNS (BruÈnger et al., 1998) followed by a simulated annealing and an individual B-factor re®nement using the complete resolution Ê , the R-factor dropped to a value of range from 30-1.6 A 32.0 %. The quality of the electron density calculated from this structure was suf®ciently high to build the remaining parts of the model. After the exchange of the complete amino acid sequence, rebuilding of the solvent model and the addition of the cofactors using the program O (Jones et al., 1991), the model was again subjected to a simulated annealing cycle. Water molecules Ê2 with isotropic temperature factors higher that 60 A were removed from the model. Iterative cycles of model building, positional re®nement and re®nement of the individual isotropic temperature factors resulted in a ®nal model with an R-factor of 16.0 % and a free R-factor Ê . As calcuof 17.9 % in the resolution range of 30-1.6 A lated with the program PROCHECK (Laskowski et al., 1993), 91.3 % of the amino acids were located in the most favored regions of the Ramachandran plot and 8.2 % in the additionally allowed regions. All of the 11 residues (0.5 %) found in the generously allowed and disallowed regions of the Ramachandran plot (not shown) were well de®ned in the electron density. Data collection, model building and refinement of the crystal structure of MCR from M. kandleri The dataset of MCR from M. kandleri was collected using a RIGAKU RAXIS 2C image plate detector mounted on a RIGAKU RU-200 rotating anode X-ray generator. Despite many efforts, only one data set with a completeness of only 65 % from a crystal of MCR from M. kanÊ resolution could be collected at dleri diffracting to 2.7 A a temperature of 90 K. The number of re¯ections corresponds to a complete dataset of a resolution of about Ê . As the quality of the collected data appeared to be 3.2 A reasonably good, the molecular replacement and re®nement were carried out with this data set. To improve the

342 data/parameter ratio, the 2-fold non-crystallographic symmetry was constrained during re®nement. Molecular replacement was carried out using the program CNS and a model of the atomic resolution structure of MCR from M. thermoautotrophicum without cofactors, solvent molecules and amino acid modi®cations. A unique solution with an R-factor of 45 % was obtained. After a simulated annealing run with the program CNS which resulted in a drop of Rcryst/Rfree to 32 %/37 %, an electron density was calculated. The quality of this electron density was suf®ciently high to model most of the side-chain conformations. After replacement of the amino acid sequence, the model of MCR from M. kandleri was subjected to cycles of interactive positional re®nement model building and grouped B-factor re®nement. The obtained values of Rcryst and Rfree (Table 1) are in the range expected for data of the quality obtained in the measurement. Accession numbers Coordinates and structure factors of MCR from M. barkeri and M. kandleri have been deposited with the Protein Data Bank, Accession codes 1e6y, r1e6ysf, 1e6v, r1e6vsf.

Acknowledgments The authors thank Hartmut Michel for generous support and the beamline staff of the EMBL Hamburg for help during the X-ray measurements. This work was supported by the Max-Planck-Gesellschaft, by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.

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Edited by R. Huber (Received 6 March 2000; received in revised form 28 August 2000; accepted 28 August 2000)