Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.     

Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.     

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.     

Crystal structure of serine dehydratase from rat liver

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.     

Role of Lys-226 in the catalytic mechanism of Bacillus stearothermophilus serine hydroxymethyltransferase--crystal structure and kinetic studies

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.     

The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase

The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the alpha-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.     

Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein

Cystathionine beta-synthase (CBS) is a unique heme- containing enzyme that catalyzes a pyridoxal 5'-phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an inherited disease of sulfur metabolism characterized by increased levels of the toxic metabolite homocysteine. Here we present the X-ray crystal structure of a truncated form of the enzyme. CBS shares the same fold with O-acetylserine sulfhydrylase but it contains an additional N-terminal heme binding site. This heme binding motif together with a spatially adjacent oxidoreductase active site motif could explain the regulation of its enzyme activity by redox changes.     

Molecular basis of cysteine biosynthesis in plants: structural and functional analysis of O-acetylserine sulfhydrylase from Arabidopsis thaliana

In plants, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment and provides the only metabolic sulfide donor for the generation of methionine, glutathione, phytochelatins, iron-sulfur clusters, vitamin cofactors, and multiple secondary metabolites. O-Acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-acetylserine into cysteine. Here we describe the 2.2 A resolution crystal structure of OASS from Arabidopsis thaliana (AtOASS) and the 2.7 A resolution structure of the AtOASS K46A mutant with PLP and methionine covalently linked as an external aldimine in the active site. Although the plant and bacterial OASS share a conserved set of amino acids for PLP binding, the structure of AtOASS reveals a difference from the bacterial enzyme in the positioning of an active site loop formed by residues 74-78 when methionine is bound. Site-directed mutagenesis, kinetic analysis, and ligand binding titrations probed the functional roles of active site residues. These experiments indicate that Asn(77) and Gln(147) are key amino acids for O-acetylserine binding and that Thr(74) and Ser(75) are involved in sulfur incorporation into cysteine. In addition, examination of the AtOASS structure and nearly 300 plant and bacterial OASS sequences suggest that the highly conserved beta8A-beta9A surface loop may be important for interaction with serine acetyltransferase, the other enzyme in cysteine biosynthesis. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis.     

The serine acetyltransferase reaction: acetyl transfer from an acylpantothenyl donor to an alcohol

Serine acetyltransferase is a member of the left-handed parallel beta-helix family of enzymes that catalyzes the committed step in the de novo synthesis of l-cysteine in bacteria and plants. The enzyme has an ordered kinetic mechanism with acetyl CoA bound prior to l-serine and O-acetyl-l-serine released prior to CoA. The rate-limiting step along the reaction pathway is the nucleophilic attack of the serine hydroxyl on the thioester of acetyl CoA. Product release contributes to rate-limitation at saturating concentrations of reactants. The reaction is catalyzed by an active site general base with a pK of 7, which accepts a proton from the serine hydroxyl as a tetrahedral intermediate is formed between the reactants, and donates it to the thiol of CoA as the intermediate collapses to give products. This mechanism is likely the same for all O-acyltransferases that catalyze their reaction by direct attack of the alcohol on the acyl donor, using an active-site histidine as the general base. Serine acetyltransferase is regulated by feedback inhibition by the end product l-cysteine, which acts by binding to the serine site in the active site and inducing a conformational change that prevents reactant binding. The enzyme also associates with O-acetylserine sulfhydrylase, the final enzyme in the biosynthetic pathway, which contributes to stabilizing the acetyltransferase.     

Identification and biochemical characterization of serine hydroxymethyl transferase in the hydrogenosome of Trichomonas vaginalis

Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.     

Structural and biochemical analyses of Microcystis aeruginosa O-acetylserine sulfhydrylases reveal a negative feedback regulation of cysteine biosynthesis

O-acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis from O-acetylserine (OAS) and inorganic sulfide in plants and bacteria. Bioinformatics analyses combined with activity assays enabled us to annotate the two putative genes of Microcystis aeruginosa PCC 7806 to CysK1 and CysK2, which encode the two 75% sequence-identical OASS paralogs. Moreover, we solved the crystal structures of CysK1 at 2.30 ? and cystine-complexed CysK2 at 1.91 ?, revealing a quite similar overall structure that belongs to the family of fold-type II PLP-dependent enzymes. Structural comparison indicated a significant induced fit upon binding to the cystine, which occupies the binding site for the substrate OAS and blocks the product release tunnel. Subsequent enzymatic assays further confirmed that cystine is a competitive inhibitor of the substrate OAS. Moreover, multiple-sequence alignment revealed that the cystine-binding residues are highly conserved in all OASS proteins, suggesting that this auto-inhibition of cystine might be a universal mechanism of cysteine biosynthesis pathway.     

Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 A resolution. CysM (Rv1336) is a PLP-containing beta-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 A resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.     

Sulfite formation by wine yeasts

Four strains of wine yeasts of two different species (Saccharomyces cerevisiae var. ellipsoideus and Saccharomyces bayanus) were investigated with respect to the influence of various sulfur compounds on the formation of O-acetylserine sulfhydrylase, O-acetylhomoserine sulfhydrylase and serine sulfhydrase. The specific enzyme activities were followed over a growth period of 96 h.In the presence of sulfate, sulfite and djencolic acid during exponential growth, a moderate increase of O-acetylserine sulfhydrylase and O-acetylhomoserine sulfhydrylase activities was recognized. In three strains cysteine and methionine prevented this derepression. At the end of the exponential growth phase, biosynthesis of these two enzymes was suppressed again. Serine sulfhydrase showed a modified regulation which indicates that its synthesis and the synthesis of O-acetylserine and O-acetylhomoserine sulfhydrylases are not coordinated.Abbreviations OAS O-acetylserine - OAHS O-acetylhomoserine     

Pyridoxal 5'-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B₆ requiring enzymes. PLP reacts with apo-B₆ enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B₆ enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B₆ proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B₆ enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.     

The CymR regulator in complex with the enzyme CysK controls cysteine metabolism in Bacillus subtilis

Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.     

Visualization of PLP-bound intermediates in hemeless variants of human cystathionine beta-synthase: evidence that lysine 119 is a general base

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to give cystathionine in a pyridoxal phosphate (PLP)-dependent reaction. The human enzyme contains a single heme per monomer that is bound in an N-terminal 69 amino acid extension that is missing from the otherwise highly homologous yeast enzyme. The heme dominates the UV-visible spectrum and obscures kinetic characterization of the PLP-bound reaction intermediates. In this study, we have engineered a hemeless mutant of human cystathionine beta-synthase by deletion of the N-terminal 69 amino acids. The resulting variant displays approximately 40% of the activity seen with the wild type enzyme, binds stoichiometric amounts of PLP, and permits spectral characterization of PLP-based intermediates. The enzyme as isolated exhibits an absorption maximum at 412nm corresponding to a protonated internal aldimine. Addition of serine shifts the lambdamax to 420nm (assigned as the external aldimine) with a broad shoulder between 450 and 500nm (assigned as the aminoacrylate intermediate). Addition of the product, cystathionine, also leads to formation of an external aldimine (420nm). Homocysteine elicits a red shift (and a decrease in absorption) in the spectrum from 412 to 424nm and an increase in absorption at 330nm, presumably due to formation of a dead-end complex. Mutation of K119, the residue that forms the Schiff base, to alanine results in a approximately 10(3)-fold decrease in activity, which increases approximately 2-fold in the presence of an exogenous base, ethylamine. Spectral shifts (412 --> 420nm) consistent with the formation of external aldimines are observed in the presence of serine or cystathionine, but an aminoacrylate intermediate is not formed at detectable levels. These results are consistent with an additional role for K119 as a general base in the reaction catalyzed by human cystathionine beta-synthase.     

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Two structures of alliinase from Alliium sativum L.: apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLP cofactor

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent alpha, beta-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the epsilon-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.     

Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme

Two classes of cystathionine beta-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine. The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown. (31)P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T(1) = 6.34 +/- 0.01 s) and the diamagnetic ferrous (T(1) = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other. This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron. However, the (31)P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket. These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory. The (31)P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes. The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates. The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.