- Email: [email protected]
The ACT domain family
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The ACT domain family David M Chipman* and Boaz Shaanan† A novel ligand-binding domain, named the ‘ACT domain’, was recently identified by a PSI-BLAST search. The archetypical ACT domain is the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH), which folds with a ferredoxin-like βαββαβ topology. A pair of ACT domains form an eight-stranded antiparallel sheet with two molecules of the allosteric inhibitor serine bound in the interface. The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand binding fold. Rat phenylalanine hydroxylase has a regulatory domain with a similar fold, but different ligand-binding mode. Putative ACT domains in some proteins of unknown structure (e.g. acetohydroxyacid synthase regulatory subunits) may also fold like the 3PGDH regulatory domain. The regulatory domain of threonine deaminase, although not a member of the ACT sequence family, is similar in structure to the paired 3PGDH regulatory domains. Repeats of ACT-like domains can create nonequivalent ligand-binding sites with the potential for complex regulatory patterns. The structures and mechanisms of such systems have only begun to be examined. Addresses Department of Life Sciences, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Structural Biology 2001, 11:694–700 0959-440X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations 3PGDH 3-phosphoglycerate dehydrogenase ACT aspartate kinase – chorismate mutase – TyrA AHAS acetohydroxyacid synthase ATCase aspartate transcarbamylase tetrahydrobiopterin BH4 DCoH bifunctional mammalian pterin-4a-carbinolamine dehydratase/dimerization cofactor of HNF1 PDB Protein Data Bank PheOH phenylalanine hydroxylase PSI-BLAST position-sensitive iterative database search rmsd root mean square deviation TD threonine deaminase
Introduction It has been recognized for decades that enzymes catalyzing different reactions with different metabolic roles often contain very similar structural motifs, related to, for example, cofactor binding, catalytic activity or interaction with nucleic acids. Only recently, however, have structurally conserved domains involved in the binding of small-molecule regulatory ligands been recognized in functionally diverse proteins. Aravind and Koonin [1•] discovered a previously unrecognized conserved sequence domain when they seeded a PSI-BLAST search (position-sensitive iterative database search) with the sequence of the Escherichia coli
IlvN protein, the 90 amino acid long regulatory subunit of acetohydroxyacid synthase (AHAS) isozyme I. Aravind and Koonin coined the name ‘ACT domain’, after three of the allosterically regulated enzymes in which this sequence domain is found: aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase). The ACT sequence domain found in E. coli 3-phosphoglycerate dehydrogenase (3PGDH) corresponds in the crystallographic structure to a clearly separated C-terminal domain that contains the binding site for the regulatory ligand, serine [2]. On the basis of these observations, Aravind and Koonin [1•,3••] suggested that the ACT domain corresponds to a regulatory ligand binding domain, with a common ligand-binding mode, that was fused into a wide variety of proteins in the course of protein evolution. Recent work on several proteins has provided experimental tests for this hypothesis. The ACT domain consensus sequence (Figure 1) has been detected in the sequences of proteins involved in amino acid and purine biosynthesis; in mammalian phenylalanine hydroxylases; in enzymes involved in the general regulation of bacterial metabolism (e.g. uridylyl transferases); and in many as yet uncharacterized open reading frames (ORFs). The metabolic roles and regulatory properties of some of these proteins are given in Table 1, together with the linear arrangement of domains in proteins from representative organisms. In proteins with ACT repeats, the conservation of the ACT consensus sequence is often much more clear for one putative domain than for others. Notably, the domain arrangements of enzymes with the same catalytic function are not necessarily conserved across species. For example, the well-characterized allosteric chorismate mutase from yeast has a regulatory domain completely unrelated to the ACT domain [4,5], whereas the chorismate mutases that do contain an ACT domain are multifunctional enzymes, generally fused to prephenate dehydratase. Such observations obviously give support to the notion that the catalytic and regulatory functions of enzymes developed independently. This review will discuss the known 3D structures of ACT domains and ‘ACT-like’ domains, as well as the biochemical evidence supporting the hypothesis that other ACT domains have similar folds. The implications of this hypothesis for the evolutionary development of varied regulatory mechanisms will also be briefly discussed.
ACT domains of known three-dimensional structure E. coli 3PGDH was the first protein with an ACT domain whose 3D structure was determined; its C-terminal regulatory domain can be considered to represent the archetypical ACT structure. This 74 amino acid domain folds as an αβ sandwich with ferredoxin-like βαββαβ topology, as shown in Figure 2 [6,7]. In the homotetrameric holoenzyme,
The ACT domain family Chipman and Shaanan
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Figure 1 Consensus sequence SS_cons SerA_Ecoli/337-408 SS PH4H_RAT/35-110 SS IlvN_Ecoli/9-81 IlvH_Ecoli/3-76 AHAS-RSU-Athal/R1 AHAS-RSU-Athal/R2 LysC_Ecoli/314-384 ThrA_Ecoli/318-384 ThrA_Ecoli/400-468 PheA_Ecoli/298-376 TyrA_Bsubt/299-371 PheA_Bsubt/201-277 PurU_Ecoli/7-79 TyrR_Ecoli/1-70 GlnD_Ecoli/815-884 GlnD_Ecoli/708-780 RelA_Ecoli/667-741
..l.h...scsGhl.pl..hhsp.shsl..h...........s...h...................b............ ceeeeeeecctthhhhhhhhhhcttccccceeeeecssccceeeeeecccg...gghhhhhhhhhhhhcttcccceeec ↓ ↓ ↓ RRLMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSA.QMGYVVIDIEAD......EDVAEKALQAMKAIPGTIRARLL ceeeeeeecctthhhhhhhhhhtttceeeeeeeeecs.seeeeeeeeecc......hhhhhhhhhhhhcstteeeeeec ISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFFTYLDK...RTKPVLGSIIKSLRNDIGATVHELS eeeeeeeecctthhhhhhhhhhcttccttseeeeecssctteeeeeecbcg...gghhhhhhhhhhhhhttcccceeee VILELTVRNHPGVMTDVCGLFARRAFNVEGILCLPIQ.DSDKSHIWLLVN.....DDQRLEQMISQIDKLEDVVKVQRN RILSVLLEN ESGALSRVIGLFSQRGYN IESLTVAPTD.DPTLSRMTIQTV....GDEKVLEQIEKQLHKLVDVLRVSEL HTISVFVGDESGMINRIAGVFARRGYNIESLAVGLNR.DK..ALFTIVVC....GTERVLQQVIEQLQK.LVNVLKVED HTLSLLVNDIPGVLNIVTGVFARRGYNIQSLAVGHAE.TKGISRITTVIP....ATDESVSKLVQQLYK.LVDVHEVHD HSLNMLHS..RGFLAEVFGILARHNISVDLITTSEVS..VALTLDTTGST..STGDTLLTQSLLMEL..SALCRVEVEE FSSGPGMKGMVGMAARVFAAMSRARISVVLITQSSSE......YSISFCVP......QSDCVRAERAMLEEFYLELKE. SVVGDGLRTLRGISAKFFAALARANINIVAIAQGSSE.....RSISVVVNN...DDATTGVRVTHQM..LFNTDQVIEV TTLLMATGQQAGALVEALLVLRNHNLIMTRLESRPIHGNPWEEMFYLDIQA..NLESAEMQKALKELGEITRSMK.... YDLYVDVPDHPGVISEITAILAAERISITNIRIIETR.EDINGILRISFQ.....SDDDRKRAEQCIEARAEYETFYAD LMVMLPQDDQSGALHRVLSAFSWRNLNLSKIESRPTK..TGLGHYFFIIDIEKAFDDVLIPGAMQELE.ALGCKVRLL. KVLRTICPDQKGLIARITNICYKHELNIVQNNEFVDH.RTGRFFMRTELE.....GIFNDSTLLADLDSALPEGSVREL MRLEVFCEDRLGLTRELLDLLVLRGIDLRGIEIDPIG.RIYLNFAELEFE........SFSSLMAEIRRIAGVTDVRTV SFLELIALDQPGLLARVGKIFADLGISLHGARITTIG..ERVEDLFIIAT....ADRRALNNELQQ...EVHQRLTEAL TEIFIWSPDRPYLFAAVCAELDRRNLSVHDAQIFTTR.DGMAMDTFIVLE....PDGNPLSADRHEVI.RFGLEQVLTQ LVVRVVANDRSGLLRDITTILANEKVNVLGVASRSDT.KQQLATIDMTIEI...YNLQVLGRVLGKLNQVPDVIDARRL
P08328 ss P04176 ss P08143 P00894 D84725 D84725 P08660 P00561 P00561 P07022 P20692 P21203 P37051 P07604 P27249 P27249 P11585
THD1_Ecoli/339-411 SS THD1_Ecoli/434-505 SS
ALLAVTIPEEKGSFLKFCQLLGGRS.VTEFNYRFADA.KNACIFVGVRLSR....GLEERKEILQMLNDGGYSVVDLSD eeeeeeccbssscshhhhhttssse.eeeeeeecccs.sbceeeeeeecss....thhhhhhhhhhhtsssceeettss RLYSFEFPESPGALLRFLNTLGTYWNISLFHYRSHGT.DYGRVLAAFELG....DHE..PDFETRLNELGYDCHDETNN eeeeeecccctthhhhhhhhhcsccccceeecbcttt.cssceeeeecxx....xxx..xxxxxxxxxxxceeeecttc
P04968 ss P04968 ss
sa_cons
Current Opinion in Structural Biology
Alignment of the amino acid sequences of representative members of the ACT domain family (Pfam accession number PF01842). The consensus sequence for this family is given in the top line: b, big amino acid (FILMVWYKREQ); c, charged amino acid (DEHKR); h, hydrophobic amino acid (ACFILMVWY); l, branched amino acid (ILV); p, polar amino acid (DEHKNQRST); s, small amino acid (ACSTDNVGP). The second line is a secondary structure consensus based on the experimental structures of E. coli 3PGDH (SerA) and rat PheOH (PH4H). The amino acids of 3PGDH that contact bound serine are shown in bold type and underlined; the three residues making direct polar contacts are indicated by arrows. Residues in other proteins whose spontaneous mutation leads to loss of the
effector response are shown in bold reversed print and residues whose directed mutation leads to a similar effect are boxed. Most of the sequences shown are the indicated segments of proteins that appear in Table 1; others are IlvN, regulatory subunit of AHAS isozyme I; AHAS-RSU-Athal, the AHAS regulatory subunit from A. thaliana, where R1 and R2 are the first and second repeats; THD1, threonine deaminase. TD is not identified by sequence as having an ACT domain. The column on the far right gives the Swiss-Prot primary accession number for the sequence. SS, the experimentally observed secondary structure for each protein: c, 310 helix; e, extended (β) strand; h, α helix; t, turn; x, amino acid not resolved in the crystallographic structure.
pairs of regulatory domains interact to form an eight-stranded β sheet with four helices on one side. 3PGDH is the only protein with an ACT domain for which the position of a bound ligand has been unambiguously determined. Serine is an allosteric inhibitor of 3PGDH that lowers the catalytic rate of the enzyme at substrate saturation without a significant effect on the apparent KM (V-type inhibition). In the crystal structure, four molecules of serine are bound to the homotetramer, each essentially buried in the subunit interfaces. Each serine makes extensive contacts with its own regulatory domain and additional contacts with the symmetry-related domain [6,7] (Figure 3). It has been suggested that the hydrogen-bonding network thus formed holds the two domains together; unfortunately, the structure of the ligand-free protein is yet to be determined.
A domain at the N terminus (residues 34–117) of rat phenylalanine hydroxylase (PheOH) is very similar to the archetypical regulatory domain of 3PGDH [8•] (Figure 2). Although the effector sites have not been localized, chemical and genetic modifications of the protein clearly indicate that this is the regulatory domain. Unlike 3PGDH, however, the PheOH regulatory domains do not interact with one another [8•]. Kobe et al. [8•] suggest that the inhibitory ligand tetrahydrobiopterin (BH4) is bound in a manner analogous to its position in a related pterin-binding protein, DCoH [9] (Figure 2). Indirect biochemical evidence suggests that the activating ligand, phenylalanine, might be bound at the interface between the regulatory and catalytic domains, near the second β strand. If these suggestions turn out to be correct, then the hypothesis that ACT domains all share a common mode of small ligand binding [1•] will have to be modified.
The amino acids in close contact with the bound serine are emphasized in the sequence in Figure 1; the fact that several of these residues are among the most conserved in the ACT consensus sequence supports the hypothesis that these domains share a common ligand-binding mode.
Threonine deaminase: ACT-like domains E. coli threonine deaminase (threonine dehydratase, TD) contains domains that possess structural, rather than
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Table 1 Some representative proteins with ACT-like domains. Typical domain structure†
Protein
Metabolic system*
Example
Regulatory ligand
3-phosphoglycerate dehydrogenase
Ser
E. coli SerA
Ser(–)
subs
Acetohydroxyacid synthase (regulatory subunit)
Val-Leu-Ile
E. coli IlvH Arabidopsis [18•]
Val(–) Ile, Val(–)
ACT
Aspartate kinase
Thr, Met and/or Lys
B. subtilis LysC
Lys(–)
aak
ACT
E. coli LysC
Lys(–)
aak
ACT
E. coli ThrA
Thr(–)
aak
ACT
ACT
pdt
ACT
Homoserine dehydrogenase
Thr and Met
nad
ACT
ACT
ACT
ACT
Chorismate mutase
Phe
E. coli PheA
Phe(–)
chm
Prephenate dehydrogenase
Tyr
B. subtilis TyrA
Tyr(–)
pdh
ACT
Phe(–)
pdt
ACT
Met(+), Gly(–)
ACT
formt bioph
Prephenate dehydratase Formyl FH4 hydrolase
Phe Purine
B. subtilis PheA E. coli PurU
Phenylalanine hydroxylase
Phe catabolism
Rat PheOH
Phe(+), BH4(–)
ACT
TyrR protein
Aromatic amino acid (transcription)
E. coli TyrR
Tyr (repression)
ACT
PII UTase/URase
Gln synthetase regulation
E. coli GlnD
Gln (R+), Glu (T+)
Ntr
ppGpp synthetase
Stringent response
E. coli RelA
Unknown
Threonine deaminase
Ile
S. typhim. IlvA
Ile(–), Val(+)
plp
ACT‡
B. subtilis IlvA
Ile(–), Val(+)
plp
ACT‡
hdh
sig54
Nhyd
ACT
ACT
ACT ACT‡
*Unless otherwise indicated, the protein is an enzyme participating in the biosynthesis of the small molecule indicated in this column. Phenylalanine hydroxylase is involved in phenylalanine catabolism (and, to some extent, tyrosine biosynthesis) in mammals. The PII protein is the uridylyl transferase/hydrolase involved in the regulatory cascade controlling glutamine synthetase in E. coli. The stringent response is a pleotropic response of metabolic pathways to starvation for charged tRNAs, mediated by the nucleotide ppGpp. †Boxes represent sequence-defined domains as
they appear in the Pfam database. In addition to the ACT domain, the 3PGDH substrate (subs); NAD- binding (nad); aspartate kinase (aak); homoserine dehydrogenase (hdh); chorismate mutase (chm); prephenate dehydratase (pdt); prephenate dehydrogenase (pdh); formyl transferase (formt); biopterin-dependent hydroxylase (bioph); sigma factor 54 (sig54); nucleotide transferase (Ntr); nucleotide hydrolase (Nhyd); pyridoxal phosphate (plp); and 'ACT-like' (ACT‡) domains are labeled. Unlabeled domains have unclear functions.
sequence, homology to the 3PGDH and PheOH regulatory domains. TD is the first enzyme on the pathway for the biosynthesis of isoleucine and was the first enzyme for which allosteric behavior was carefully characterized [10]. The effectors of TD modulate the apparent affinity for substrate without significant influence on the rate at substrate saturation (K-type regulation). The regulatory behavior of TD has been successfully explained in terms of an expanded two-state Monod-Wyman-Changeux (MWC) concerted allosteric model [11], in which the physiological effectors isoleucine (an inhibitor) and valine (a positive effector) compete. Like 3PGDH, the holoenzyme is a homotetramer in which the intersubunit contacts lie between pairs of C-terminal regulatory domains and pairs of N-terminal domains [12]. In this case, however, each regulatory domain itself folds as an eight-stranded antiparallel sheet, which can be divided into two half-sheets related by a pseudo-twofold, with helices on one side (Figure 2). Gallagher et al. [12] noted a striking similarity between the structure of this domain and that of the dimer of regulatory domains in 3PGDH.
database site (http://www.sanger.ac.uk/Software/Pfam/) classifies them as belonging to a separate sequence family of TD regulatory domains. The close structural and functional relationship between these regulatory structures can not be ignored, however, and any consideration of the evolutionary implications of the ACT domain must include TD regulatory domains. Indeed, the SCOP (Structural Classification Of Proteins) database classifies the regulatory domains of 3PGDH, TD and PheOH together as the “regulatory domain in amino acid metabolism” family. Perhaps the regulatory domains of TD should thus be called ‘ACT-like’ domains.
The sequences of the regulatory half-domains of TD are not assigned by PSI-BLAST as belonging to the ACT family (see bottom four lines of Figure 1). Also, the Pfam
As certain mutations in the second half-domain (colorless, left half-domain in Figure 2) of E. coli TD lead to proteins with immeasurably low affinities for isoleucine and valine [12,13], it was suggested that only one of two potential sites in the pseudosymmetrical TD structure [12] (labeled 2 in Figure 2) is an effector-binding site. On the other hand, recent studies on Arabidopsis thaliana TD [14••], a plant enzyme similar in sequence to E. coli TD, considered the possibility that each monomer contains a pair of effectorbinding sites, equivalent to sites 1 and 2 in Figure 2. Tyr→Leu mutations were introduced in the first or second repeats of A. thaliana TD and both the equilibrium binding and allosteric effects of isoleucine and valine were examined.
The ACT domain family Chipman and Shaanan
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Figure 3
Figure 2
(a)
N
(b)
N
C
?
C
Asn346 Asn364′ Serine
WW
His344
C N Thr352 (c)
(d)
N
N Current Opinion in Structural Biology
1
C
2 C (e)
N
C
Current Opinion in Structural Biology
Ribbon diagrams of ACT domains of known structure and some related proteins, all from the same viewpoint, after optimal superposition of mainchain atoms on a single regulatory domain of 3PGDH, using the program ALIGN [29]. Homologous secondary structure elements of the superimposed structures are colored red, yellow, green, blue, purple and orange in order of sequence; additional structural elements are shown in gray. Ligands detected by crystallography are shown as ball-and-stick models and hypothetical ligand-binding sites are marked by ovals. (a) A dimer of 3PGDH regulatory domains with symmetry-related bound serines (PDB entry 1PSD [2,7]). (b) Regulatory domain of rat PheOH (PDB entry 1PHZ [8•]; rmsd of Cα atoms after superposition 2.2 Å). (c) Regulatory domain of TD (PDB entry 1TDJ [12]; rmsd 1.8 Å). Gray elements correspond to the ‘second repeat’. Note that a segment between the last two strands is not observed in the crystallographic structure. (d) The bifunctional pterin dehydratase DCoH — a signal transduction protein with a bound pterin derivative (PDB entry 1DCP [9]; rmsd 2.6 Å). Gray elements are a coil and N-terminal helix that have no correspondence in 3PGDH. (e) N-terminal domain of an ATCase regulatory subunit, with bound CTP (PDB entry 5AT1 [30]; rmsd 2.6 Å). The gray fifth strand has no correspondence in 3PGDH. The drawing was prepared with the program MOLSCRIPT [31].
No simple explanation involving a single site in a monomer could be found for the results of these experiments. It was therefore suggested that site 1 (Figure 2) represents a highaffinity isoleucine-binding site, whereas the other site binds isoleucine at lower affinity, leading to inhibition [14••]. Valine competing with isoleucine at site 1 would lead to a state in which the enzyme does not bind effectors at site 2.
The serine-binding region of 3PGDH. The loop between the first strand and the following helix of one domain (right) has two residues that make direct polar contacts with serine (Asn346 and His344). A third such contact is provided by a group at the end of the first helix of the other domain (Asn364′). The view is from the opposite side of the domain from that shown in Figure 2, but the color scheme has been retained. A strand from the first domain that would cover the serine in this view is not shown. The drawing was prepared with MOLSCRIPT, using coordinates from PDB entry 1PSD [7].
It is significant that there are other TDs, for example, that of Bacillus subtilis, that have only a single ACT-like domain in a protomer. Like the E. coli and A. thaliana enzymes, this protein is inhibited by isoleucine, whereas valine competes with isoleucine and counters its effect [15]. As Gallagher et al. [12] point out, these forms of TD might assemble in a manner similar to the larger TDs, resulting in unpaired ACT-like domains with four-stranded β sheets, or rather might associate so that their regulatory domains form symmetric eight-stranded domains resembling those of 3PGDH. The intrinsic difference between proteins with single ACT-like domains and those with two (or more) ACT-like repeats is, of course, that the latter potentially have nonequivalent and therefore independently evolving ligand-binding sites.
Biochemical evidence concerning ACT domains Biochemical exploration of a few other proteins containing ACT sequence domains supports the suggestion that these domains contain the archetypical ACT structure. Bacterial AHAS, the first common enzyme on the pathway leading to formation of the branched-chain amino acids, usually shows feedback inhibition by valine, one of the end products of the pathway. Separate regulatory subunits are necessary for valine sensitivity and full activity of the catalytic subunits. E. coli AHAS isozyme III shows mixed V- and K-type inhibition by valine, with substantial residual activity remaining at valine saturation. Mendel et al. [16••] addressed the structure of the valine-binding region of the regulatory subunits by the selection of spontaneous valineresistant mutants and construction of additional directed mutants. A hybrid fold-recognition algorithm [17] predicted with a high confidence level that the domain could fold
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like the 3PGDH regulatory domain. The properties of more than a dozen mutant forms of the AHAS III regulatory subunit (Figure 1) lend support to a model in which valine interacts with amino acids in positions homologous to those that interact with serine in the dimerized regulatory domains of 3PGDH ([16••]; S Mendel, personal communication). Plant AHASs are generally sensitive to all three of the branched-chain amino acids. The two AHAS regulatory subunits that have been cloned from plants contain large internal repeats [18•,19], each repeat starting with an ACT domain. The A. thaliana enzyme reconstituted with the complete regulatory subunit shows synergistic inhibition by valine and leucine, whereas the enzyme reconstituted with a construct containing only the first repeat is inhibited by leucine alone [18•]. Accordingly, Lee and Duggleby suggest that the A. thaliana AHAS regulatory subunits have a structure in which two ACT repeats with sites of slightly different specificity interact with one another, as suggested for the plant TD. For E. coli aspartate kinase III (the LysC protein), both mutational analyses [20] (Figure 1) and fold prediction support a fold and ligand-binding mode for the regulatory domains that are similar to those for 3PGDH. Functionally related aspartate kinases have either single ACT domains or repeats (Table 1); as in the cases discussed above, the pairs of nonequivalent domains may have similar sites, two very different sites or share a single ligand-binding site. It is interesting to note that, whereas one of the two ACT domains of the E. coli ThrA product (aspartate kinase/homoserine dehydrogenase) is most compatible with the 3PGDH fold, the second is predicted to be more compatible with several other possible folds [17].
Domains that may be related to ACT Several other protein domains have intriguing structural similarities to the ACT domain. The bifunctional mammalian pterin-4a-carbinolamine dehydratase/dimerization cofactor of HNF1 (DCoH) is a small, single-domain protein with αββαββα topology [9]; the 60 residues of the βαββα motif are folded very much like the equivalent (i.e. N-terminal) part of the PheOH regulatory domain (Figure 2). A superposition of these parts of the proteins has an rmsd of 1.81 Å for Cα atoms [8•]. The position of the bound inhibitor 7,8-dihydrobiopterin, on which Kobe et al. [8•] based their proposal for BH4 binding to PheOH, is very different from the position of serine in 3PGDH. Another domain with a possible relationship to the ACT domain is the N-terminal domain of the regulatory subunit of aspartate transcarbamylase (ATCase) [21], the well-studied, nucleotide-regulated first committed enzyme of pyrimidine biosynthesis (Figure 2). A pair of these domains from two protomers interact to form a continuous antiparallel sheet, but, in this case, an additional β strand completes each domain, so that the extended sheet contains ten strands. The regulatory ligand binding site in ATCase, however,
is unrelated to the sites in the other proteins we have considered (Figure 2) and is distant from the domain interface.
Mechanisms of regulation The effects of ligands on proteins with ACT-like regulatory domains vary. Some proteins are inhibited, a few (e.g. PheOH) are activated by the regulatory ligand, whereas others (e.g. TD) have competing inhibitory and activating modulators of physiological significance. Some show K-type allosteric effects, which fit the MWC model, whereas others (e.g. 3PGDH and AHAS) have V-type effects. Tyrosine binding to the TyrR protein represses transcription of a group of operons and effector binding to the bifunctional regulatory uridylyl transferase/hydrolase (PII UTase/URase) alters the relative rates of the two different reactions that it catalyzes. E. coli 3PGDH is the only case for which the detailed mechanism for the transduction of the effect of modulator binding is beginning to be understood. The effects of serine on 3PGDH have been studied by direct binding measurements, spectroscopic studies and site-directed mutagenesis [6,7,22–25,26••]. Substrate binding to 3PGDH is noncooperative and the KM for phosphoglycerate is affected very little by serine. Substrate association and product release, rather than chemistry, appear to dominate the turnover rate of the enzyme. The enzyme binds its first two serine molecules with positive cooperativity, whereas the next two bind with greatly decreased affinity. The binding of a single serine at a regulatory subunit dimerization interface is assumed to close the domain interface [22,23] (Figure 2). This is proposed to lead to a hinge-like movement of the regulatory domain relative to the substrate-binding domain, resulting in the locking of the active site cleft (between the substrate- and nucleotide-binding domains) in an open and inactive conformation. As it has been impossible to obtain stable crystals of 3PGDH in the absence of serine, it has been necessary to study the mechanism of transduction by mutagenesis and modeling. In a recent series of papers, Grant’s group at Washington University [26••,27,28•] has described the investigation of various domain–domain interactions. Their studies suggest that, following serine binding at one regulatory domain interface, separate pathways transmit the effects to the active site cleft and to the other regulatory domain interface.
Conclusions The recognition of the common and widespread sequence homology among a group of small domains found in a variety of proteins that are regulated by small-molecule effectors raises many interesting questions. To date, the structures of only two of these ACT-like domains have been determined (the regulatory domains of 3PGDH and PheOH) and, although they display similarity, it would be premature to conclude that all of the sequences detected by the PSI-BLAST search have similar 3D structures. There certainly is currently no firm support for the hypothesis that the ligand-binding modes of different ACT-like
The ACT domain family Chipman and Shaanan
domains are similar. In cases in which a threading algorithm supports the fold prediction based on the distant sequence homology, further experimental tests of ligand-binding sites (e.g. [16••]) can provide useful information. On the other hand, consideration of TD suggests that a protein domain can have a 3D structure much like members of the ACT domain family without being detected by a PSIBLAST search. Is the αβ sandwich so common and stable a structure that domains with a particular topology may represent completely independent inventions? In order to take the idea of a common ACT-like ligand-binding structure beyond the realm of interesting hypothesis, further structural and ligand-binding studies on additional proteins are needed. Such studies will no doubt reveal many new variations on the structural theme of the ACT domain.
Acknowledgements The original work from our laboratories described here was supported by grant 243/98 from the Israel Science Foundation to DMC and Z Barak. DMC is the incumbent of the Lily and Sidney Oelbaum Chair in Applied Biochemistry.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1. •
Aravind L, Koonin EV: Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. J Mol Biol 1999, 287:1023-1040. The authors investigated the ability of the PSI-BLAST iterative search method to detect nonobvious relationships among proteins. It is assumed that sequence relationships imply structural similarity, so this method should aid in making structural predictions. In addition to uncovering the ACT domain family, several other relationships are uncovered: the connection of a family of metalloproteases to proteins with the HSP70-actin fold, a new superfamily of immunoglobulin-like domains and so on. 2.
Schuller DJ, Grant GA, Banaszak LJ: The allosteric ligand site in the V-max-type cooperative enzyme phosphoglycerate dehydrogenase. Nat Struct Biol 1995, 2:69-76.
3. ••
Anantharaman V, Koonin EV, Aravind L: Regulatory potential, phyletic distribution and evolution of ancient, intracellular smallmolecule-binding domains. J Mol Biol 2001, 307:1271-1292. Starting from the observation that distinct compact small-molecule-binding domains (SMBDs) play roles in many types of regulation, the authors carried out a comprehensive examination of the occurrence of such domains in the sequence databases. Three new SMBD families were detected, but the major thrust of the paper is the cataloging of the diversity of domain architectures and functions for SMBDs, and a discussion of the evolutionary implications of this information. It is a fascinating overview, but some readers may not agree that such far reaching conclusions can be made on the basis of sequence analyses without further structural studies.
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regulatory domain, with structural similarity to that of 3PGDH, flexibly linked to the catalytic domain. The similarity of this domain to DCoH is also noted. Phosphorylation has no major structural effects and the interaction between covalent and allosteric regulation is discussed. There is still little experimental support for the proposed ligand-binding sites. 9.
Cronk JD, Endrizzi JA, Alber T: High-resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue. Protein Sci 1996, 5:1963-1972.
10. Changeux JP: The feedback control mechanism of biosynthetic L-threonine deaminase by L-isoleucine. Cold Spring Harb Symp Quant Biol 1961, 26:313-330. 11. Eisenstein E, Yu HD, Fisher KE, Lacuzio DA, Ducote KR, Schwarz FP: An expanded two-state model accounts for homotropic cooperativity in biosynthetic threonine deaminase from Escherichia coli. Biochemistry 1995, 34:9403-9412. 12. Gallagher DT, Gilliland GL, Xiao G, Zondlo J, Fisher KE, Chinchilla D, Eisenstein E: Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 1998, 6:465-475. 13. Chinchilla D, Schwarz FP, Eisenstein E: Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. J Biol Chem 1998, 273:23219-23224. 14. Wessel PM, Graciet E, Douce R, Dumas R: Evidence for two distinct •• effector binding sites in threonine deaminase by site-directed mutagenesis, kinetic and binding experiments. Biochemistry 2000, 39:15136-15143. The conclusions of this study on the A. thaliana TD enzyme are very different from those reached in Eisenstein’s thorough studies on the E. coli enzyme [11–13]. In this case, there is strong evidence for two allosteric ligand sites with different ligand affinities and effects on catalytic activity. The model proposed is interesting food for thought and illustrates the potential complexities of a protein with repeats of an ACT-like domain. 15. Hatfield GW, Umbarger HE: Threonine deaminase from Bacillus subtilis II. The steady state kinetic properties. J Biol Chem 1970, 245:1742-1747. 16. Mendel S, Elkayam T, Sella C, Vyazmensky M, Chipman DM, Barak Z: •• Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis. J Mol Biol 2001, 307:465-477. A fold recognition approach suggests that the N-terminal part of AHAS regulatory subunits might be structurally homologous to the 3PGDH regulatory domains. Kinetics, genetics and site-directed mutagenesis identify amino acids involved in ligand interactions and provide support for the idea that AHAS regulatory domains form a ligand-binding interface like that of 3PGDH. 17.
Fischer D: Hybrid fold recognition: combining sequence derived properties with evolutionary information. In Pacific Symposium on Biocomputing 2000. Edited by Altman RB, Dunker K, Hunter L, Lauderdale K, Klein TE. Singapore: World Scientific; 2000:119-130.
4.
Xue Y, Lipscomb WN, Graf R, Schnappauf G, Braus G: The crystal structure of allosteric chorismate mutase at 2.2-Å resolution. Proc Natl Acad Sci USA 1994, 91:10814-10818.
18. Lee YT, Duggleby RG: Identification of the regulatory subunit of • Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit. Biochemistry 2001, 40:6836-6844. The ideas of Wessel et al. [14••] concerning TD are applied to a plant AHAS regulatory subunit, for which there is clear evidence of a sequence repeat. The synergistic interaction of allosteric effectors, together with the properties of individual repeat segments, could imply that the quaternary arrangement of this enzyme is different from that of the bacterial enzymes [16••].
5.
Strater N, Schnappauf G, Braus G, Lipscomb WN: Mechanisms of catalysis and allosteric regulation of yeast chorismate mutase from crystal structures. Structure 1997, 5:1437-1452.
19. Hershey HP, Schwartz LJ, Gale JP, Abell LM: Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia. Plant Mol Biol 1999, 40:795-806.
6.
Al Rabiee R, Zhang Y, Grant GA: The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase: site-directed mutagenesis of effector binding site residues. J Biol Chem 1996, 271:23235-23238.
20. Kikuchi Y, Kojima H, Tanaka T: Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli. FEMS Microbiol Lett 1999, 173:211-215.
7.
Grant GA, Schuller DJ, Banaszak LJ: A model for the regulation of D-3-phosphoglycerate dehydrogenase, a V-max-type allosteric enzyme. Protein Sci 1996, 5:34-41.
8. •
Kobe B, Jennings IG, House CM, Michell BJ, Goodwill KE, Santarsiero BD, Stevens RC, Cotton RGH, Kemp BE: Structural basis of autoregulation of phenylalanine hydroxylase. Nat Struct Biol 1999, 6:442-448. The crystallographic structures of phosphorylated and dephosphorylated dimeric forms of rat PheOH are presented. The structures reveal a separate
21. Honzatko RB, Crawford JL, Monaco HL, Ladner JE, Ewards BF, Evans DR, Warren SG, Wiley DC, Ladner RC, Lipscomb WN: Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli. J Mol Biol 1982, 160:219-263. 22. Al Rabiee R, Lee EJ, Grant GA: The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase: cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding. J Biol Chem 1996, 271:13013-13017.
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Catalysis and regulation
23. Grant GA, Xu XL: Probing the regulatory domain interface of D-3-phosphoglycerate dehydrogenase with engineered tryptophan residues. J Biol Chem 1998, 273:22389-22394.
27.
24. Grant GA, Xu XL, Hu ZQ: The relationship between effector binding and inhibition of activity in D-3-phosphoglycerate dehydrogenase. Protein Sci 1999, 8:2501-2505.
28. Grant GA, Hu Z, Xu XL: Amino acid residue mutations uncouple • cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase. J Biol Chem 2001, 276:17844-17850. Although this paper does not even deal with the regulatory domain per se, it is an impressive attempt to understand the regulatory mechanism by mutagenesis of individual residues and analysis of the possible conformational changes they could be involved in. This is the latest in an important series of papers by this group on 3PGDH.
25. Grant GA, Xu XL, Hu ZQ: Removal of the tryptophan 139 side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative effects. Arch Biochem Biophys 2000, 375:171-174. 26. Grant GA, Hu ZQ, Xu XL: Specific interactions at the regulatory •• domain-substrate binding domain interface influence the cooperativity of inhibition and effector binding in Escherichia coli D-3-phosphoglycerate dehydrogenase. J Biol Chem 2001, 276:1078-1083. Mutagenesis of a number of residues that participate in hydrogen bonding across the regulatory domain–substrate-binding domain interfaces leads to changes in the kinetic parameters of 3PGDH. These changes are analyzed in the context of the crystallographic structure and the assumption that rigid movements of domains are involved. The only available structure is of the enzyme fully occupied by four serine (inhibitor) molecules, so the mutagenesis approach has been crucial to understanding regulation.
Grant GA, Xu XL, Hu Z: Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli D-3-phosphoglycerate dehydrogenase. Biochemistry 2000, 39:7316-7319.
29. Cohen GH: ALIGN: a program to superimpose protein coordinates, accounting for insertions and deletions. J Appl Crystallogr 1997, 30:1160-1161. 30. Gouaux JE, Stevens RC, Lipscomb WN: Crystal structures of aspartate carbamoyltransferase ligated with phosphonoacetamide, malonate, and CTP or ATP at 2.8-Å resolution and neutral pH. Biochemistry 1990, 29:7702-7715. 31. Kraulis PJ: MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991, 24:946-950.
The ACT domain family David M Chipman* and Boaz Shaanan† A novel ligand-binding domain, named the ‘ACT domain’, was recently identified by a PSI-BLAST search. The archetypical ACT domain is the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH), which folds with a ferredoxin-like βαββαβ topology. A pair of ACT domains form an eight-stranded antiparallel sheet with two molecules of the allosteric inhibitor serine bound in the interface. The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand binding fold. Rat phenylalanine hydroxylase has a regulatory domain with a similar fold, but different ligand-binding mode. Putative ACT domains in some proteins of unknown structure (e.g. acetohydroxyacid synthase regulatory subunits) may also fold like the 3PGDH regulatory domain. The regulatory domain of threonine deaminase, although not a member of the ACT sequence family, is similar in structure to the paired 3PGDH regulatory domains. Repeats of ACT-like domains can create nonequivalent ligand-binding sites with the potential for complex regulatory patterns. The structures and mechanisms of such systems have only begun to be examined. Addresses Department of Life Sciences, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Structural Biology 2001, 11:694–700 0959-440X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations 3PGDH 3-phosphoglycerate dehydrogenase ACT aspartate kinase – chorismate mutase – TyrA AHAS acetohydroxyacid synthase ATCase aspartate transcarbamylase tetrahydrobiopterin BH4 DCoH bifunctional mammalian pterin-4a-carbinolamine dehydratase/dimerization cofactor of HNF1 PDB Protein Data Bank PheOH phenylalanine hydroxylase PSI-BLAST position-sensitive iterative database search rmsd root mean square deviation TD threonine deaminase
Introduction It has been recognized for decades that enzymes catalyzing different reactions with different metabolic roles often contain very similar structural motifs, related to, for example, cofactor binding, catalytic activity or interaction with nucleic acids. Only recently, however, have structurally conserved domains involved in the binding of small-molecule regulatory ligands been recognized in functionally diverse proteins. Aravind and Koonin [1•] discovered a previously unrecognized conserved sequence domain when they seeded a PSI-BLAST search (position-sensitive iterative database search) with the sequence of the Escherichia coli
IlvN protein, the 90 amino acid long regulatory subunit of acetohydroxyacid synthase (AHAS) isozyme I. Aravind and Koonin coined the name ‘ACT domain’, after three of the allosterically regulated enzymes in which this sequence domain is found: aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase). The ACT sequence domain found in E. coli 3-phosphoglycerate dehydrogenase (3PGDH) corresponds in the crystallographic structure to a clearly separated C-terminal domain that contains the binding site for the regulatory ligand, serine [2]. On the basis of these observations, Aravind and Koonin [1•,3••] suggested that the ACT domain corresponds to a regulatory ligand binding domain, with a common ligand-binding mode, that was fused into a wide variety of proteins in the course of protein evolution. Recent work on several proteins has provided experimental tests for this hypothesis. The ACT domain consensus sequence (Figure 1) has been detected in the sequences of proteins involved in amino acid and purine biosynthesis; in mammalian phenylalanine hydroxylases; in enzymes involved in the general regulation of bacterial metabolism (e.g. uridylyl transferases); and in many as yet uncharacterized open reading frames (ORFs). The metabolic roles and regulatory properties of some of these proteins are given in Table 1, together with the linear arrangement of domains in proteins from representative organisms. In proteins with ACT repeats, the conservation of the ACT consensus sequence is often much more clear for one putative domain than for others. Notably, the domain arrangements of enzymes with the same catalytic function are not necessarily conserved across species. For example, the well-characterized allosteric chorismate mutase from yeast has a regulatory domain completely unrelated to the ACT domain [4,5], whereas the chorismate mutases that do contain an ACT domain are multifunctional enzymes, generally fused to prephenate dehydratase. Such observations obviously give support to the notion that the catalytic and regulatory functions of enzymes developed independently. This review will discuss the known 3D structures of ACT domains and ‘ACT-like’ domains, as well as the biochemical evidence supporting the hypothesis that other ACT domains have similar folds. The implications of this hypothesis for the evolutionary development of varied regulatory mechanisms will also be briefly discussed.
ACT domains of known three-dimensional structure E. coli 3PGDH was the first protein with an ACT domain whose 3D structure was determined; its C-terminal regulatory domain can be considered to represent the archetypical ACT structure. This 74 amino acid domain folds as an αβ sandwich with ferredoxin-like βαββαβ topology, as shown in Figure 2 [6,7]. In the homotetrameric holoenzyme,
The ACT domain family Chipman and Shaanan
695
Figure 1 Consensus sequence SS_cons SerA_Ecoli/337-408 SS PH4H_RAT/35-110 SS IlvN_Ecoli/9-81 IlvH_Ecoli/3-76 AHAS-RSU-Athal/R1 AHAS-RSU-Athal/R2 LysC_Ecoli/314-384 ThrA_Ecoli/318-384 ThrA_Ecoli/400-468 PheA_Ecoli/298-376 TyrA_Bsubt/299-371 PheA_Bsubt/201-277 PurU_Ecoli/7-79 TyrR_Ecoli/1-70 GlnD_Ecoli/815-884 GlnD_Ecoli/708-780 RelA_Ecoli/667-741
..l.h...scsGhl.pl..hhsp.shsl..h...........s...h...................b............ ceeeeeeecctthhhhhhhhhhcttccccceeeeecssccceeeeeecccg...gghhhhhhhhhhhhcttcccceeec ↓ ↓ ↓ RRLMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSA.QMGYVVIDIEAD......EDVAEKALQAMKAIPGTIRARLL ceeeeeeecctthhhhhhhhhhtttceeeeeeeeecs.seeeeeeeeecc......hhhhhhhhhhhhcstteeeeeec ISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFFTYLDK...RTKPVLGSIIKSLRNDIGATVHELS eeeeeeeecctthhhhhhhhhhcttccttseeeeecssctteeeeeecbcg...gghhhhhhhhhhhhhttcccceeee VILELTVRNHPGVMTDVCGLFARRAFNVEGILCLPIQ.DSDKSHIWLLVN.....DDQRLEQMISQIDKLEDVVKVQRN RILSVLLEN ESGALSRVIGLFSQRGYN IESLTVAPTD.DPTLSRMTIQTV....GDEKVLEQIEKQLHKLVDVLRVSEL HTISVFVGDESGMINRIAGVFARRGYNIESLAVGLNR.DK..ALFTIVVC....GTERVLQQVIEQLQK.LVNVLKVED HTLSLLVNDIPGVLNIVTGVFARRGYNIQSLAVGHAE.TKGISRITTVIP....ATDESVSKLVQQLYK.LVDVHEVHD HSLNMLHS..RGFLAEVFGILARHNISVDLITTSEVS..VALTLDTTGST..STGDTLLTQSLLMEL..SALCRVEVEE FSSGPGMKGMVGMAARVFAAMSRARISVVLITQSSSE......YSISFCVP......QSDCVRAERAMLEEFYLELKE. SVVGDGLRTLRGISAKFFAALARANINIVAIAQGSSE.....RSISVVVNN...DDATTGVRVTHQM..LFNTDQVIEV TTLLMATGQQAGALVEALLVLRNHNLIMTRLESRPIHGNPWEEMFYLDIQA..NLESAEMQKALKELGEITRSMK.... YDLYVDVPDHPGVISEITAILAAERISITNIRIIETR.EDINGILRISFQ.....SDDDRKRAEQCIEARAEYETFYAD LMVMLPQDDQSGALHRVLSAFSWRNLNLSKIESRPTK..TGLGHYFFIIDIEKAFDDVLIPGAMQELE.ALGCKVRLL. KVLRTICPDQKGLIARITNICYKHELNIVQNNEFVDH.RTGRFFMRTELE.....GIFNDSTLLADLDSALPEGSVREL MRLEVFCEDRLGLTRELLDLLVLRGIDLRGIEIDPIG.RIYLNFAELEFE........SFSSLMAEIRRIAGVTDVRTV SFLELIALDQPGLLARVGKIFADLGISLHGARITTIG..ERVEDLFIIAT....ADRRALNNELQQ...EVHQRLTEAL TEIFIWSPDRPYLFAAVCAELDRRNLSVHDAQIFTTR.DGMAMDTFIVLE....PDGNPLSADRHEVI.RFGLEQVLTQ LVVRVVANDRSGLLRDITTILANEKVNVLGVASRSDT.KQQLATIDMTIEI...YNLQVLGRVLGKLNQVPDVIDARRL
P08328 ss P04176 ss P08143 P00894 D84725 D84725 P08660 P00561 P00561 P07022 P20692 P21203 P37051 P07604 P27249 P27249 P11585
THD1_Ecoli/339-411 SS THD1_Ecoli/434-505 SS
ALLAVTIPEEKGSFLKFCQLLGGRS.VTEFNYRFADA.KNACIFVGVRLSR....GLEERKEILQMLNDGGYSVVDLSD eeeeeeccbssscshhhhhttssse.eeeeeeecccs.sbceeeeeeecss....thhhhhhhhhhhtsssceeettss RLYSFEFPESPGALLRFLNTLGTYWNISLFHYRSHGT.DYGRVLAAFELG....DHE..PDFETRLNELGYDCHDETNN eeeeeecccctthhhhhhhhhcsccccceeecbcttt.cssceeeeecxx....xxx..xxxxxxxxxxxceeeecttc
P04968 ss P04968 ss
sa_cons
Current Opinion in Structural Biology
Alignment of the amino acid sequences of representative members of the ACT domain family (Pfam accession number PF01842). The consensus sequence for this family is given in the top line: b, big amino acid (FILMVWYKREQ); c, charged amino acid (DEHKR); h, hydrophobic amino acid (ACFILMVWY); l, branched amino acid (ILV); p, polar amino acid (DEHKNQRST); s, small amino acid (ACSTDNVGP). The second line is a secondary structure consensus based on the experimental structures of E. coli 3PGDH (SerA) and rat PheOH (PH4H). The amino acids of 3PGDH that contact bound serine are shown in bold type and underlined; the three residues making direct polar contacts are indicated by arrows. Residues in other proteins whose spontaneous mutation leads to loss of the
effector response are shown in bold reversed print and residues whose directed mutation leads to a similar effect are boxed. Most of the sequences shown are the indicated segments of proteins that appear in Table 1; others are IlvN, regulatory subunit of AHAS isozyme I; AHAS-RSU-Athal, the AHAS regulatory subunit from A. thaliana, where R1 and R2 are the first and second repeats; THD1, threonine deaminase. TD is not identified by sequence as having an ACT domain. The column on the far right gives the Swiss-Prot primary accession number for the sequence. SS, the experimentally observed secondary structure for each protein: c, 310 helix; e, extended (β) strand; h, α helix; t, turn; x, amino acid not resolved in the crystallographic structure.
pairs of regulatory domains interact to form an eight-stranded β sheet with four helices on one side. 3PGDH is the only protein with an ACT domain for which the position of a bound ligand has been unambiguously determined. Serine is an allosteric inhibitor of 3PGDH that lowers the catalytic rate of the enzyme at substrate saturation without a significant effect on the apparent KM (V-type inhibition). In the crystal structure, four molecules of serine are bound to the homotetramer, each essentially buried in the subunit interfaces. Each serine makes extensive contacts with its own regulatory domain and additional contacts with the symmetry-related domain [6,7] (Figure 3). It has been suggested that the hydrogen-bonding network thus formed holds the two domains together; unfortunately, the structure of the ligand-free protein is yet to be determined.
A domain at the N terminus (residues 34–117) of rat phenylalanine hydroxylase (PheOH) is very similar to the archetypical regulatory domain of 3PGDH [8•] (Figure 2). Although the effector sites have not been localized, chemical and genetic modifications of the protein clearly indicate that this is the regulatory domain. Unlike 3PGDH, however, the PheOH regulatory domains do not interact with one another [8•]. Kobe et al. [8•] suggest that the inhibitory ligand tetrahydrobiopterin (BH4) is bound in a manner analogous to its position in a related pterin-binding protein, DCoH [9] (Figure 2). Indirect biochemical evidence suggests that the activating ligand, phenylalanine, might be bound at the interface between the regulatory and catalytic domains, near the second β strand. If these suggestions turn out to be correct, then the hypothesis that ACT domains all share a common mode of small ligand binding [1•] will have to be modified.
The amino acids in close contact with the bound serine are emphasized in the sequence in Figure 1; the fact that several of these residues are among the most conserved in the ACT consensus sequence supports the hypothesis that these domains share a common ligand-binding mode.
Threonine deaminase: ACT-like domains E. coli threonine deaminase (threonine dehydratase, TD) contains domains that possess structural, rather than
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Catalysis and regulation
Table 1 Some representative proteins with ACT-like domains. Typical domain structure†
Protein
Metabolic system*
Example
Regulatory ligand
3-phosphoglycerate dehydrogenase
Ser
E. coli SerA
Ser(–)
subs
Acetohydroxyacid synthase (regulatory subunit)
Val-Leu-Ile
E. coli IlvH Arabidopsis [18•]
Val(–) Ile, Val(–)
ACT
Aspartate kinase
Thr, Met and/or Lys
B. subtilis LysC
Lys(–)
aak
ACT
E. coli LysC
Lys(–)
aak
ACT
E. coli ThrA
Thr(–)
aak
ACT
ACT
pdt
ACT
Homoserine dehydrogenase
Thr and Met
nad
ACT
ACT
ACT
ACT
Chorismate mutase
Phe
E. coli PheA
Phe(–)
chm
Prephenate dehydrogenase
Tyr
B. subtilis TyrA
Tyr(–)
pdh
ACT
Phe(–)
pdt
ACT
Met(+), Gly(–)
ACT
formt bioph
Prephenate dehydratase Formyl FH4 hydrolase
Phe Purine
B. subtilis PheA E. coli PurU
Phenylalanine hydroxylase
Phe catabolism
Rat PheOH
Phe(+), BH4(–)
ACT
TyrR protein
Aromatic amino acid (transcription)
E. coli TyrR
Tyr (repression)
ACT
PII UTase/URase
Gln synthetase regulation
E. coli GlnD
Gln (R+), Glu (T+)
Ntr
ppGpp synthetase
Stringent response
E. coli RelA
Unknown
Threonine deaminase
Ile
S. typhim. IlvA
Ile(–), Val(+)
plp
ACT‡
B. subtilis IlvA
Ile(–), Val(+)
plp
ACT‡
hdh
sig54
Nhyd
ACT
ACT
ACT ACT‡
*Unless otherwise indicated, the protein is an enzyme participating in the biosynthesis of the small molecule indicated in this column. Phenylalanine hydroxylase is involved in phenylalanine catabolism (and, to some extent, tyrosine biosynthesis) in mammals. The PII protein is the uridylyl transferase/hydrolase involved in the regulatory cascade controlling glutamine synthetase in E. coli. The stringent response is a pleotropic response of metabolic pathways to starvation for charged tRNAs, mediated by the nucleotide ppGpp. †Boxes represent sequence-defined domains as
they appear in the Pfam database. In addition to the ACT domain, the 3PGDH substrate (subs); NAD- binding (nad); aspartate kinase (aak); homoserine dehydrogenase (hdh); chorismate mutase (chm); prephenate dehydratase (pdt); prephenate dehydrogenase (pdh); formyl transferase (formt); biopterin-dependent hydroxylase (bioph); sigma factor 54 (sig54); nucleotide transferase (Ntr); nucleotide hydrolase (Nhyd); pyridoxal phosphate (plp); and 'ACT-like' (ACT‡) domains are labeled. Unlabeled domains have unclear functions.
sequence, homology to the 3PGDH and PheOH regulatory domains. TD is the first enzyme on the pathway for the biosynthesis of isoleucine and was the first enzyme for which allosteric behavior was carefully characterized [10]. The effectors of TD modulate the apparent affinity for substrate without significant influence on the rate at substrate saturation (K-type regulation). The regulatory behavior of TD has been successfully explained in terms of an expanded two-state Monod-Wyman-Changeux (MWC) concerted allosteric model [11], in which the physiological effectors isoleucine (an inhibitor) and valine (a positive effector) compete. Like 3PGDH, the holoenzyme is a homotetramer in which the intersubunit contacts lie between pairs of C-terminal regulatory domains and pairs of N-terminal domains [12]. In this case, however, each regulatory domain itself folds as an eight-stranded antiparallel sheet, which can be divided into two half-sheets related by a pseudo-twofold, with helices on one side (Figure 2). Gallagher et al. [12] noted a striking similarity between the structure of this domain and that of the dimer of regulatory domains in 3PGDH.
database site (http://www.sanger.ac.uk/Software/Pfam/) classifies them as belonging to a separate sequence family of TD regulatory domains. The close structural and functional relationship between these regulatory structures can not be ignored, however, and any consideration of the evolutionary implications of the ACT domain must include TD regulatory domains. Indeed, the SCOP (Structural Classification Of Proteins) database classifies the regulatory domains of 3PGDH, TD and PheOH together as the “regulatory domain in amino acid metabolism” family. Perhaps the regulatory domains of TD should thus be called ‘ACT-like’ domains.
The sequences of the regulatory half-domains of TD are not assigned by PSI-BLAST as belonging to the ACT family (see bottom four lines of Figure 1). Also, the Pfam
As certain mutations in the second half-domain (colorless, left half-domain in Figure 2) of E. coli TD lead to proteins with immeasurably low affinities for isoleucine and valine [12,13], it was suggested that only one of two potential sites in the pseudosymmetrical TD structure [12] (labeled 2 in Figure 2) is an effector-binding site. On the other hand, recent studies on Arabidopsis thaliana TD [14••], a plant enzyme similar in sequence to E. coli TD, considered the possibility that each monomer contains a pair of effectorbinding sites, equivalent to sites 1 and 2 in Figure 2. Tyr→Leu mutations were introduced in the first or second repeats of A. thaliana TD and both the equilibrium binding and allosteric effects of isoleucine and valine were examined.
The ACT domain family Chipman and Shaanan
697
Figure 3
Figure 2
(a)
N
(b)
N
C
?
C
Asn346 Asn364′ Serine
WW
His344
C N Thr352 (c)
(d)
N
N Current Opinion in Structural Biology
1
C
2 C (e)
N
C
Current Opinion in Structural Biology
Ribbon diagrams of ACT domains of known structure and some related proteins, all from the same viewpoint, after optimal superposition of mainchain atoms on a single regulatory domain of 3PGDH, using the program ALIGN [29]. Homologous secondary structure elements of the superimposed structures are colored red, yellow, green, blue, purple and orange in order of sequence; additional structural elements are shown in gray. Ligands detected by crystallography are shown as ball-and-stick models and hypothetical ligand-binding sites are marked by ovals. (a) A dimer of 3PGDH regulatory domains with symmetry-related bound serines (PDB entry 1PSD [2,7]). (b) Regulatory domain of rat PheOH (PDB entry 1PHZ [8•]; rmsd of Cα atoms after superposition 2.2 Å). (c) Regulatory domain of TD (PDB entry 1TDJ [12]; rmsd 1.8 Å). Gray elements correspond to the ‘second repeat’. Note that a segment between the last two strands is not observed in the crystallographic structure. (d) The bifunctional pterin dehydratase DCoH — a signal transduction protein with a bound pterin derivative (PDB entry 1DCP [9]; rmsd 2.6 Å). Gray elements are a coil and N-terminal helix that have no correspondence in 3PGDH. (e) N-terminal domain of an ATCase regulatory subunit, with bound CTP (PDB entry 5AT1 [30]; rmsd 2.6 Å). The gray fifth strand has no correspondence in 3PGDH. The drawing was prepared with the program MOLSCRIPT [31].
No simple explanation involving a single site in a monomer could be found for the results of these experiments. It was therefore suggested that site 1 (Figure 2) represents a highaffinity isoleucine-binding site, whereas the other site binds isoleucine at lower affinity, leading to inhibition [14••]. Valine competing with isoleucine at site 1 would lead to a state in which the enzyme does not bind effectors at site 2.
The serine-binding region of 3PGDH. The loop between the first strand and the following helix of one domain (right) has two residues that make direct polar contacts with serine (Asn346 and His344). A third such contact is provided by a group at the end of the first helix of the other domain (Asn364′). The view is from the opposite side of the domain from that shown in Figure 2, but the color scheme has been retained. A strand from the first domain that would cover the serine in this view is not shown. The drawing was prepared with MOLSCRIPT, using coordinates from PDB entry 1PSD [7].
It is significant that there are other TDs, for example, that of Bacillus subtilis, that have only a single ACT-like domain in a protomer. Like the E. coli and A. thaliana enzymes, this protein is inhibited by isoleucine, whereas valine competes with isoleucine and counters its effect [15]. As Gallagher et al. [12] point out, these forms of TD might assemble in a manner similar to the larger TDs, resulting in unpaired ACT-like domains with four-stranded β sheets, or rather might associate so that their regulatory domains form symmetric eight-stranded domains resembling those of 3PGDH. The intrinsic difference between proteins with single ACT-like domains and those with two (or more) ACT-like repeats is, of course, that the latter potentially have nonequivalent and therefore independently evolving ligand-binding sites.
Biochemical evidence concerning ACT domains Biochemical exploration of a few other proteins containing ACT sequence domains supports the suggestion that these domains contain the archetypical ACT structure. Bacterial AHAS, the first common enzyme on the pathway leading to formation of the branched-chain amino acids, usually shows feedback inhibition by valine, one of the end products of the pathway. Separate regulatory subunits are necessary for valine sensitivity and full activity of the catalytic subunits. E. coli AHAS isozyme III shows mixed V- and K-type inhibition by valine, with substantial residual activity remaining at valine saturation. Mendel et al. [16••] addressed the structure of the valine-binding region of the regulatory subunits by the selection of spontaneous valineresistant mutants and construction of additional directed mutants. A hybrid fold-recognition algorithm [17] predicted with a high confidence level that the domain could fold
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Catalysis and regulation
like the 3PGDH regulatory domain. The properties of more than a dozen mutant forms of the AHAS III regulatory subunit (Figure 1) lend support to a model in which valine interacts with amino acids in positions homologous to those that interact with serine in the dimerized regulatory domains of 3PGDH ([16••]; S Mendel, personal communication). Plant AHASs are generally sensitive to all three of the branched-chain amino acids. The two AHAS regulatory subunits that have been cloned from plants contain large internal repeats [18•,19], each repeat starting with an ACT domain. The A. thaliana enzyme reconstituted with the complete regulatory subunit shows synergistic inhibition by valine and leucine, whereas the enzyme reconstituted with a construct containing only the first repeat is inhibited by leucine alone [18•]. Accordingly, Lee and Duggleby suggest that the A. thaliana AHAS regulatory subunits have a structure in which two ACT repeats with sites of slightly different specificity interact with one another, as suggested for the plant TD. For E. coli aspartate kinase III (the LysC protein), both mutational analyses [20] (Figure 1) and fold prediction support a fold and ligand-binding mode for the regulatory domains that are similar to those for 3PGDH. Functionally related aspartate kinases have either single ACT domains or repeats (Table 1); as in the cases discussed above, the pairs of nonequivalent domains may have similar sites, two very different sites or share a single ligand-binding site. It is interesting to note that, whereas one of the two ACT domains of the E. coli ThrA product (aspartate kinase/homoserine dehydrogenase) is most compatible with the 3PGDH fold, the second is predicted to be more compatible with several other possible folds [17].
Domains that may be related to ACT Several other protein domains have intriguing structural similarities to the ACT domain. The bifunctional mammalian pterin-4a-carbinolamine dehydratase/dimerization cofactor of HNF1 (DCoH) is a small, single-domain protein with αββαββα topology [9]; the 60 residues of the βαββα motif are folded very much like the equivalent (i.e. N-terminal) part of the PheOH regulatory domain (Figure 2). A superposition of these parts of the proteins has an rmsd of 1.81 Å for Cα atoms [8•]. The position of the bound inhibitor 7,8-dihydrobiopterin, on which Kobe et al. [8•] based their proposal for BH4 binding to PheOH, is very different from the position of serine in 3PGDH. Another domain with a possible relationship to the ACT domain is the N-terminal domain of the regulatory subunit of aspartate transcarbamylase (ATCase) [21], the well-studied, nucleotide-regulated first committed enzyme of pyrimidine biosynthesis (Figure 2). A pair of these domains from two protomers interact to form a continuous antiparallel sheet, but, in this case, an additional β strand completes each domain, so that the extended sheet contains ten strands. The regulatory ligand binding site in ATCase, however,
is unrelated to the sites in the other proteins we have considered (Figure 2) and is distant from the domain interface.
Mechanisms of regulation The effects of ligands on proteins with ACT-like regulatory domains vary. Some proteins are inhibited, a few (e.g. PheOH) are activated by the regulatory ligand, whereas others (e.g. TD) have competing inhibitory and activating modulators of physiological significance. Some show K-type allosteric effects, which fit the MWC model, whereas others (e.g. 3PGDH and AHAS) have V-type effects. Tyrosine binding to the TyrR protein represses transcription of a group of operons and effector binding to the bifunctional regulatory uridylyl transferase/hydrolase (PII UTase/URase) alters the relative rates of the two different reactions that it catalyzes. E. coli 3PGDH is the only case for which the detailed mechanism for the transduction of the effect of modulator binding is beginning to be understood. The effects of serine on 3PGDH have been studied by direct binding measurements, spectroscopic studies and site-directed mutagenesis [6,7,22–25,26••]. Substrate binding to 3PGDH is noncooperative and the KM for phosphoglycerate is affected very little by serine. Substrate association and product release, rather than chemistry, appear to dominate the turnover rate of the enzyme. The enzyme binds its first two serine molecules with positive cooperativity, whereas the next two bind with greatly decreased affinity. The binding of a single serine at a regulatory subunit dimerization interface is assumed to close the domain interface [22,23] (Figure 2). This is proposed to lead to a hinge-like movement of the regulatory domain relative to the substrate-binding domain, resulting in the locking of the active site cleft (between the substrate- and nucleotide-binding domains) in an open and inactive conformation. As it has been impossible to obtain stable crystals of 3PGDH in the absence of serine, it has been necessary to study the mechanism of transduction by mutagenesis and modeling. In a recent series of papers, Grant’s group at Washington University [26••,27,28•] has described the investigation of various domain–domain interactions. Their studies suggest that, following serine binding at one regulatory domain interface, separate pathways transmit the effects to the active site cleft and to the other regulatory domain interface.
Conclusions The recognition of the common and widespread sequence homology among a group of small domains found in a variety of proteins that are regulated by small-molecule effectors raises many interesting questions. To date, the structures of only two of these ACT-like domains have been determined (the regulatory domains of 3PGDH and PheOH) and, although they display similarity, it would be premature to conclude that all of the sequences detected by the PSI-BLAST search have similar 3D structures. There certainly is currently no firm support for the hypothesis that the ligand-binding modes of different ACT-like
The ACT domain family Chipman and Shaanan
domains are similar. In cases in which a threading algorithm supports the fold prediction based on the distant sequence homology, further experimental tests of ligand-binding sites (e.g. [16••]) can provide useful information. On the other hand, consideration of TD suggests that a protein domain can have a 3D structure much like members of the ACT domain family without being detected by a PSIBLAST search. Is the αβ sandwich so common and stable a structure that domains with a particular topology may represent completely independent inventions? In order to take the idea of a common ACT-like ligand-binding structure beyond the realm of interesting hypothesis, further structural and ligand-binding studies on additional proteins are needed. Such studies will no doubt reveal many new variations on the structural theme of the ACT domain.
Acknowledgements The original work from our laboratories described here was supported by grant 243/98 from the Israel Science Foundation to DMC and Z Barak. DMC is the incumbent of the Lily and Sidney Oelbaum Chair in Applied Biochemistry.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1. •
Aravind L, Koonin EV: Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. J Mol Biol 1999, 287:1023-1040. The authors investigated the ability of the PSI-BLAST iterative search method to detect nonobvious relationships among proteins. It is assumed that sequence relationships imply structural similarity, so this method should aid in making structural predictions. In addition to uncovering the ACT domain family, several other relationships are uncovered: the connection of a family of metalloproteases to proteins with the HSP70-actin fold, a new superfamily of immunoglobulin-like domains and so on. 2.
Schuller DJ, Grant GA, Banaszak LJ: The allosteric ligand site in the V-max-type cooperative enzyme phosphoglycerate dehydrogenase. Nat Struct Biol 1995, 2:69-76.
3. ••
Anantharaman V, Koonin EV, Aravind L: Regulatory potential, phyletic distribution and evolution of ancient, intracellular smallmolecule-binding domains. J Mol Biol 2001, 307:1271-1292. Starting from the observation that distinct compact small-molecule-binding domains (SMBDs) play roles in many types of regulation, the authors carried out a comprehensive examination of the occurrence of such domains in the sequence databases. Three new SMBD families were detected, but the major thrust of the paper is the cataloging of the diversity of domain architectures and functions for SMBDs, and a discussion of the evolutionary implications of this information. It is a fascinating overview, but some readers may not agree that such far reaching conclusions can be made on the basis of sequence analyses without further structural studies.
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regulatory domain, with structural similarity to that of 3PGDH, flexibly linked to the catalytic domain. The similarity of this domain to DCoH is also noted. Phosphorylation has no major structural effects and the interaction between covalent and allosteric regulation is discussed. There is still little experimental support for the proposed ligand-binding sites. 9.
Cronk JD, Endrizzi JA, Alber T: High-resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue. Protein Sci 1996, 5:1963-1972.
10. Changeux JP: The feedback control mechanism of biosynthetic L-threonine deaminase by L-isoleucine. Cold Spring Harb Symp Quant Biol 1961, 26:313-330. 11. Eisenstein E, Yu HD, Fisher KE, Lacuzio DA, Ducote KR, Schwarz FP: An expanded two-state model accounts for homotropic cooperativity in biosynthetic threonine deaminase from Escherichia coli. Biochemistry 1995, 34:9403-9412. 12. Gallagher DT, Gilliland GL, Xiao G, Zondlo J, Fisher KE, Chinchilla D, Eisenstein E: Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 1998, 6:465-475. 13. Chinchilla D, Schwarz FP, Eisenstein E: Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. J Biol Chem 1998, 273:23219-23224. 14. Wessel PM, Graciet E, Douce R, Dumas R: Evidence for two distinct •• effector binding sites in threonine deaminase by site-directed mutagenesis, kinetic and binding experiments. Biochemistry 2000, 39:15136-15143. The conclusions of this study on the A. thaliana TD enzyme are very different from those reached in Eisenstein’s thorough studies on the E. coli enzyme [11–13]. In this case, there is strong evidence for two allosteric ligand sites with different ligand affinities and effects on catalytic activity. The model proposed is interesting food for thought and illustrates the potential complexities of a protein with repeats of an ACT-like domain. 15. Hatfield GW, Umbarger HE: Threonine deaminase from Bacillus subtilis II. The steady state kinetic properties. J Biol Chem 1970, 245:1742-1747. 16. Mendel S, Elkayam T, Sella C, Vyazmensky M, Chipman DM, Barak Z: •• Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis. J Mol Biol 2001, 307:465-477. A fold recognition approach suggests that the N-terminal part of AHAS regulatory subunits might be structurally homologous to the 3PGDH regulatory domains. Kinetics, genetics and site-directed mutagenesis identify amino acids involved in ligand interactions and provide support for the idea that AHAS regulatory domains form a ligand-binding interface like that of 3PGDH. 17.
Fischer D: Hybrid fold recognition: combining sequence derived properties with evolutionary information. In Pacific Symposium on Biocomputing 2000. Edited by Altman RB, Dunker K, Hunter L, Lauderdale K, Klein TE. Singapore: World Scientific; 2000:119-130.
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18. Lee YT, Duggleby RG: Identification of the regulatory subunit of • Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit. Biochemistry 2001, 40:6836-6844. The ideas of Wessel et al. [14••] concerning TD are applied to a plant AHAS regulatory subunit, for which there is clear evidence of a sequence repeat. The synergistic interaction of allosteric effectors, together with the properties of individual repeat segments, could imply that the quaternary arrangement of this enzyme is different from that of the bacterial enzymes [16••].
5.
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7.
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8. •
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23. Grant GA, Xu XL: Probing the regulatory domain interface of D-3-phosphoglycerate dehydrogenase with engineered tryptophan residues. J Biol Chem 1998, 273:22389-22394.
27.
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28. Grant GA, Hu Z, Xu XL: Amino acid residue mutations uncouple • cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase. J Biol Chem 2001, 276:17844-17850. Although this paper does not even deal with the regulatory domain per se, it is an impressive attempt to understand the regulatory mechanism by mutagenesis of individual residues and analysis of the possible conformational changes they could be involved in. This is the latest in an important series of papers by this group on 3PGDH.
25. Grant GA, Xu XL, Hu ZQ: Removal of the tryptophan 139 side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative effects. Arch Biochem Biophys 2000, 375:171-174. 26. Grant GA, Hu ZQ, Xu XL: Specific interactions at the regulatory •• domain-substrate binding domain interface influence the cooperativity of inhibition and effector binding in Escherichia coli D-3-phosphoglycerate dehydrogenase. J Biol Chem 2001, 276:1078-1083. Mutagenesis of a number of residues that participate in hydrogen bonding across the regulatory domain–substrate-binding domain interfaces leads to changes in the kinetic parameters of 3PGDH. These changes are analyzed in the context of the crystallographic structure and the assumption that rigid movements of domains are involved. The only available structure is of the enzyme fully occupied by four serine (inhibitor) molecules, so the mutagenesis approach has been crucial to understanding regulation.
Grant GA, Xu XL, Hu Z: Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli D-3-phosphoglycerate dehydrogenase. Biochemistry 2000, 39:7316-7319.
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