Structure and Mechanism of O-Acetylserine Sulfhydrylase

Abstract

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a ␤-replacement reaction in which the ␤-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 ␣-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 C␤-O bond cleavage than for C␣-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 ␤-hydroxyl with a thiol in two steps. Serine acetyltransferase (EC 2.3.1.30) catalyzes the acetylation (by acetyl-CoA) of the ␤-hydroxyl of L-serine to give O-acetyl-L-serine (OAS) 1 (1). The final step, the ␣,␤-elimination of acetate from OAS and the addition of H 2 S to give L-cysteine is then catalyzed by Oacetylserine 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 (2). The A and B isozymes are homodimeric with M r of about 68,900 and 64,000, respectively, and each has one tightly bound pyridoxal 5Ј-phosphate (PLP) per subunit. The structure and mechanism of the A isozyme of OASS from S. typhimurium will be the focus of this minireview. For a more comprehensive review, see Ref.3. It has recently been shown that OASS-A is negatively regulated by small anions binding to an allosteric site at the dimer interface (4), but because of space limitations this aspect will not be discussed in this article. Kinetic MechanismThe kinetic mechanism of OASS is Ping Pong Bi Bi (Fig. 2) as shown by initial velocity studies in the absence and presence of products and dead end inhibitors, isotope exchange at equilibrium, and equilibrium spectral studies (5, 6). O-Acetyl-L-serine binds to the internal aldimine form of the enzyme (E), and acetate is released as the first product. Bisulfide then adds as the second substrate to the ␣-aminoacrylate intermediate form of the enzyme (F), and L-cysteine is released as the final product. The initial velocity pattern obtained in the absence of added inhibitors exhibits competitive inhibition by both substrates, which is normally diagnostic for a ping-pong mechanism, resulting ...

Cysteine Is Not the Sulfur Source for Iron-Sulfur Cluster and Methionine Biosynthesis in the Methanogenic Archaeon Methanococcus maripaludis

“…Second, enteric bacteria and plants synthesize cysteine via direct sulfhydrylation of O-acetylserine by O-acetylserine sulfhydrylase (40), and sulfide is the physiological sulfur donor for this reaction with K m values in the micromolar range (41,42). In contrast, even though sulfide is presumably present at high levels in methanococci, it is probably not the direct sulfur donor for cysteine biosynthesis.…”

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“…Detailed studies of StOASS provide a structural model for the reaction catalyzed by the enzyme (45). Crystal structures of uncomplexed (open) and complexed (closed) StOASS formed the basis of this model (24,25).…”

Molecular Biology, Biochemistry and Cellular Physiology of Cysteine Metabolism inArabidopsis thaliana

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