Structure and Mechanism of O-Acetylserine Sulfhydrylase

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a β-replacement reaction in which the β-acetoxy group of O-acetyl-l-serine (OAS) is replaced by bisulfide to give l-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable α-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5′-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cβ–O bond cleavage than for Cα–H bond cleavage. The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a β-replacement reaction in which the β-acetoxy group of O-acetyl-l-serine (OAS) is replaced by bisulfide to give l-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable α-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5′-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cβ–O bond cleavage than for Cα–H bond cleavage. The biosynthesis of l-cysteine in enteric bacteria, such as Salmonella typhimurium and Escherichia coli, and in plants proceeds via a two-step pathway (Fig. 1). The amino acid precursor of l-cysteine is l-serine, which undergoes a substitution of its β-hydroxyl with a thiol in two steps. Serine acetyltransferase (EC 2.3.1.30) catalyzes the acetylation (by acetyl-CoA) of the β-hydroxyl of l-serine to give O-acetyl-l-serine (OAS) 1The abbreviations used are: OAS, O-acetyl-l-serine; OASS, O-acetylserine sulfhydrylase; PLP, pyridoxal 5′-phosphate; TNB, 5-thio-2-nitrobenzoate.1The abbreviations used are: OAS, O-acetyl-l-serine; OASS, O-acetylserine sulfhydrylase; PLP, pyridoxal 5′-phosphate; TNB, 5-thio-2-nitrobenzoate. (1Kredich N.M. J. Biol. Chem. 1971; 246: 3474-3484Abstract Full Text PDF PubMed Google Scholar). The final step, the α,β-elimination of acetate from OAS and the addition of H2S to give l-cysteine is then catalyzed by O-acetylserine sulfhydrylase (OASS; EC 4.2.99.8). In enteric bacteria, two isozymes of OASS, A and B, are produced under aerobic and anaerobic growth conditions, respectively (2Mino K. Ishikawa K. J. Bacteriol. 2003; 185: 2277-2284Crossref PubMed Google Scholar). 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Google Scholar). is as the for the kinetic is with a of in the transition an anti-E2 are with an asynchronous transition state in which bond between the of and the of the OAS Schiff and bond cleavage between and the The biosynthesis of l-cysteine in enteric bacteria, such as Salmonella typhimurium and Escherichia coli, and in plants proceeds via a two-step pathway (Fig. 1). The amino acid precursor of l-cysteine is l-serine, which undergoes a substitution of its β-hydroxyl with a thiol in two steps. Serine acetyltransferase (EC 2.3.1.30) catalyzes the acetylation (by acetyl-CoA) of the β-hydroxyl of l-serine to give O-acetyl-l-serine (OAS) 1The abbreviations used are: OAS, O-acetyl-l-serine; OASS, O-acetylserine sulfhydrylase; PLP, pyridoxal 5′-phosphate; TNB, 5-thio-2-nitrobenzoate.1The abbreviations used are: OAS, O-acetyl-l-serine; OASS, O-acetylserine sulfhydrylase; PLP, pyridoxal 5′-phosphate; TNB, 5-thio-2-nitrobenzoate. (1Kredich N.M. J. Biol. Chem. 1971; 246: 3474-3484Abstract Full Text PDF PubMed Google Scholar). The final step, the α,β-elimination of acetate from OAS and the addition of H2S to give l-cysteine is then catalyzed by O-acetylserine sulfhydrylase (OASS; EC 4.2.99.8). In enteric bacteria, two isozymes of OASS, A and B, are produced under aerobic and anaerobic growth conditions, respectively (2Mino K. Ishikawa K. J. Bacteriol. 2003; 185: 2277-2284Crossref PubMed Google Scholar). The A and isozymes are with of and and each one bound pyridoxal 5′-phosphate (PLP) per subunit. The and mechanism of the A of OASS from typhimurium be the of a Biol. is by to an site the J. Biol. PubMed Google of be in kinetic mechanism of OASS is (Fig. as by in the and of and and J. Biol. Chem. 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