Molecular Evolution of Threonine Dehydratase in Bacteria

Threonine dehydratase converts L-threonine to 2-ketobutyrate. Several threonine dehydratases exist in bacteria, but their origins and evolutionary pathway are unknown. Here we analyzed all the available threonine dehydratases in bacteria and proposed an evolutionary pathway leading to the genes encoding three different threonine dehydratases CTD, BTD1 and BTD2. The ancestral threonine dehydratase might contain only a catalytic domain, but one or two ACT-like subdomains were fused during the evolution, resulting BTD1 and BTD2, respectively. Horizontal gene transfer, gene fusion, gene duplication, and gene deletion may occur during the evolution of this enzyme. The results are important for understanding the functions of various threonine dehydratases found in bacteria.     

Allosteric regulation of Bacillus subtilis threonine deaminase, a biosynthetic threonine deaminase with a single regulatory domain

The enzyme threonine deaminase (TD) is a key regulatory enzyme in the pathway for the biosynthesis of isoleucine. TD is inhibited by its end product, isoleucine, and this effect is countered by valine, the product of a competing biosynthetic pathway. Sequence and structure analyses have revealed that the protomers of many TDs have C-terminal regulatory domains, composed of two ACT-like subdomains, which bind isoleucine and valine, while others have regulatory domains of approximately half the length, composed of only a single ACT-like domain. The regulatory responses of TDs from both long and short sequence varieties appear to have many similarities, but there are significant differences. We describe here the allosteric properties of Bacillus subtilis TD ( bsTD), which belongs to the short variety of TD sequences. We also examine the effects of several mutations in the regulatory domain on the kinetics of the enzyme and its response to effectors. The behavior of bsTD can be analyzed and rationalized using a modified Monod-Wyman-Changeux model. This analysis suggests that isoleucine is a negative effector, and valine is a very weak positive effector, but that at high concentrations valine inhibits activity by competing with threonine for binding to the active site. The behavior of bsTD is contrasted with the allosteric behavior reported for TDs from Escherichia coli and Arabidopsis thaliana, TDs with two subdomains. We suggest a possible evolutionary pathway to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety, e.g., E. coli TD.     

Subdomain II of α-Isopropylmalate Synthase Is Essential for Activity: INFERRING A MECHANISM OF FEEDBACK INHIBITION*

The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product l-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.     

DNA stabilization at the Bacillus subtilis PolX core��a binding model to coordinate polymerase, AP-endonuclease and 3��-5�� exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.     

Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.     

Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations

The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric complexes with ADP-Mg(2+) as a product-bound state, and with AMPPNP-Mg(2+) as an ATP-like bound state. The structure of GlcV consists of a typical ABC-ATPase domain, comprising two subdomains, connected by a linker region to a C-terminal domain of unknown function. Comparisons of the nucleotide-free and nucleotide-bound structures of GlcV reveal re-orientations of the ABCalpha subdomain and the C-terminal domain relative to the ABCalpha/beta subdomain, and switch-like rearrangements in the P-loop and Q-loop regions. Additionally, large conformational differences are observed between the GlcV structures and those of other ABC-ATPases, further emphasizing the inherent flexibility of these proteins. Notably, a comparison of the monomeric AMPPNP-Mg(2+)-bound GlcV structure with that of the dimeric ATP-Na(+)-bound LolD-E171Q mutant reveals a +/-20 degrees rigid body re-orientation of the ABCalpha subdomain relative to the ABCalpha/beta subdomain, accompanied by a local conformational difference in the Q-loop. We propose that these differences represent conformational changes that may have a role in the mechanism of energy-transduction and/or allosteric control of the ABC-ATPase activity in bacterial importers.     

Allosteric Activation and Contrasting Properties of l-Serine Dehydratase Types 1 and 2

Bacterial l-serine dehydratases differ from mammalian l- and d-serine dehydratases and bacterial d-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 d-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between l-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with l-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of l-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.     

Evidence for subdomain flexibility in Drosophila melanogaster acetylcholinesterase

The catalytic domain of the acetylcholinesterases is composed of a single polypeptide chain, the folding of which determines two subdomains. We have linked these two subdomains by mutating two residues, I327 and D375, to cysteines, to form a disulfide bridge. As a consequence, the hydrodynamic radius of the protein was reduced, suggesting that there is some flexibility in the subdomain connection. In addition to the smaller size, the mutated protein is more stable than the wild-type protein. Therefore, the flexibility between the two domains is a weak point in terms of protein stability. As expected from the location of the disulfide bond at the rim of the active site, the kinetic studies show that it affects interactions with peripheral ligands and the entrance of some of the bulkier substrates, like o-nitrophenyl acetate. In addition, the mutations affect the catalytic step for o-nitrophenyl acetate and phosphorylation by organophosphates, suggesting that this movement between the two subdomains is connected with the cooperativity between the peripheral and catalytic sites.     

Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion

The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.     

Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.     

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.     

NMR solution structure of the RED subdomain of the Sleeping Beauty transposase

DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi‐domain protein containing the catalytic and the DNA‐binding domains. The DNA‐binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.     

Involvement of Both Dockerin Subdomains in Assembly of the Clostridium thermocellum Cellulosome

Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

Structure of an SspH1-PKN1 Complex Reveals the Basis for Host Substrate Recognition and Mechanism of Activation for a Bacterial E3 Ubiquitin Ligase

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.     

Myosin V attachment to cargo requires the tight association of two functional subdomains

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle-specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.     

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis,implicated in developmental processes

PrkC was shown to be a eukaryotic-like (Hanks-type) protein kinase from Bacillus subtilis with a structural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF beta or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane-spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co-transcribed with prkC, is also required for normal biofilm and spore formation.     

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.     

Sequence similarities between tryptophan synthase beta subunit and other pyridoxal-phosphate-dependent enzymes

On the basis of 8 tryptophan synthase beta subunits (EC 4.2.1.20) consensus patterns were constructed comprising two conserved motifs. Screening of the SWISSPROT protein sequence database with these patterns indicates similarities with O-acetylserine sulfhydrolases (EC 4.2.99.8), threonine synthases (EC 4.2.99.2), L- and D-serine dehydratases (EC 4.2.1.13/EC 4.2.1.14) and threonine dehydratases (EC 4.2.1.16). Using multiple alignment procedures the similar regions could be extended. In connection with their pyridoxal-phosphate-binding-capacity and their positions in biochemical pathways evolutionary relationships among these enzymes are discussed.