Cysteine synthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternative cysteine biosynthesis pathway in mycobacteria

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5'-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-angstroms resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602-11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.     

Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium.

S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A.     

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.     

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

O-phospho-L-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis

The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.     

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.     

Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in l-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0A resolution

O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-wavelength anomalous dispersion. A monomer consists of three domains, including an N-terminal domain with a new alpha/beta fold. The topology folds of the middle and C-terminal domains were similar to those of the O-acetylserine sulfhydrylase-A from Salmonella typhimurium and the cystathionine beta-synthase from human. The cofactor, pyridoxal 5'-phosphate, is bound in a cleft between the middle and C-terminal domains through a covalent linkage to Lys127. Based on the structure determined, O-phospho-l-serine could be rationally modeled into the active site of the enzyme. An enzyme-substrate complex model and a mutation experiment revealed that Arg297, unique to hyperthermophilic archaea, is one of the most crucial residues for O-phosphoserine sulfhydrylation activity. There are more hydrophobic areas and less electric charges at the dimer interface, compared to the S.typhimurium O-acetylserine sulfhydrylase.     

Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana

In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by β-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.     

Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.     

Methionine Biosynthesis in Lemna: STUDIES ON THE REGULATION OF CYSTATHIONINE gamma-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE

Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine gamma-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine gamma-synthase, it was observed that (a) adding external methionine (2 mum) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 muml-aminoethoxyvinylglycine or with 36 mum lysine plus 4 mum threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine gamma-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase.The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine gamma-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine gamma-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O-acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis.     

Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase

Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.     

Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.     

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.     

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.     

Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1

L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.     

A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants

Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.     

A single change of histidine to glutamine alters the substrate preference of a stilbene synthase.

Stilbene and chalcone synthases are related polyketide synthases which use the same substrates but form different products. The environment of the condensing active site cysteine is highly conserved, except for the positions -2 and -3. All chalcone synthases contain Gln-Gln and prefer 4-coumaroyl-CoA as starter CoA ester, while the two known stilbene synthases contain Gln-His or His-Gln (preference phenylpropionyl-CoA and 4-coumaroyl-CoA, respectively). We investigated whether the presence and/or position of the histidine influences the substrate preference and the product specificity (stilbene or chalcone). The two amino acid motifs in the chalcone synthase from Pinus sylvestris (Gln-Gln) and in the stilbene synthases from P. sylvestris (Gln-His) and Arachis hypogaea (His-Gln) were changed by site-directed mutagenesis into all sequence combinations as found in the natural enzymes. Assays with the mutant proteins showed that the histidine does not determine the product specificity. With the chalcone and the stilbene synthase from P. sylvestris, any sequence deviation reduced the activity without marked effects on the substrate preference. The stilbene synthase from A. hypogaea was different. The change from His-Gln to Gln-His abolished enzyme activity almost completely with all three substrates. The change to Gln-Gln selectively reduced the activity with 4-coumaroyl-CoA, and the kinetic analysis indicated a slight increase in Km and a 3-fold reduction of Vmax, when compared with the parent enzyme. This converted the enzyme from a resveratrol-forming into a dihydropinosylvin-forming stilbene synthase.     

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.     

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.