Identification of the Gene Encoding Sulfopyruvate Decarboxylase, an Enzyme Involved in Biosynthesis of Coenzyme M

Abstract

The products of two adjacent genes in the chromosome of Methanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in Streptomyces wedmorensis. These two M. jannaschii genes were recombinantly expressed in Escherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of ␣-ketoacids. Both subunits are required to form an ␣ 6 ␤ 6 dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The M. jannaschii sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.Coenzyme M (2-mercaptoethanesulfonic acid) was originally characterized as one of several coenzymes involved in the formation of methane in the methanoarchaea (14). Recently it has been shown to function as a coenzyme in the bacterial metabolism of aliphatic alkenes (1). Despite the fact that the pathway for its biosynthesis (Fig. 1) has been known for a number of years (35-37), no genes or enzymes involved in its biosynthesis have been identified. One of the steps in the biosynthesis of coenzyme M, the decarboxylation of sulfopyruvate to sulfoacetaldehyde, is chemically very analogous to the decarboxylation of phosphonopyruvate that occurs in the biosynthesis of natural products containing a C-P bond. Among these are fosfomycin (19), phosphinothricin (30), and bialaphos (24), each produced by various species of Streptomyces. The sequence homology among the Streptomyces genes for phosphonopyruvate decarboxylase (30), the genes contained in Methanococcus jannaschii MJ0256 (7), and the Methanobacterium thermoautotrophicum genes MT1206 and MT1207 (31) (Fig. 2) has prompted us to clone and overexpress the two proteins encoded by the MJ0256 open reading frame. This work has established that these two protein products catalyze the decarboxylation of sulfopyruvate to form sulfoacetaldehyde and CO 2 . The enzyme has been named sulfopyruvate decarboxylase, and the gene has been named comDE, to indicate that the enzymatic reaction is the fourth expected in the biosynthesis of coenzyme M from phosphoenol pyruvate and bisulfite. From sequence comparisons with other similar thiamine-PP (TPP)-dependent enzymes (Fig. 2) and from the nature of the reaction catalyzed, it is clear that this enzyme is a TPP-dependent enzyme since it contains the TPP-binding motif (DGDGSILMNLGSLSTIGYMNPKNYILVIIDN) (18). Unexpectedly, the enzyme was found to be readily inactivated by exposure to oxygen. MATERIALS AND METHODSChemicals. Sulfopyruvate and sulfoacetaldehyde were prepared as previously described (36, 37). Zhibing Lu, Department of Chemistry, University of New Mex...

Automated metabolic reconstruction for Methanococcus jannaschii

“…However, since detection of this activity, it has been possible to show that spermidine biosynthesis in Gram-positive bacteria and in archaea is similar to the related pathway in Gram-negative bacteria and in eukarya (Sekowska et al 2000). Finally, MJCyc provides strong evidence for the presence of the pathway for biosynthesis of Coenzyme M, an important cofactor in methanogenesis: the first two reactions have been identified in M. jannaschii and attributed to this pathway (MJ0255, 2r-phospho-3-sulfolactate synthase and MJ1140, 2-phosphosulfolactate phosphatase) (Graupner et al 2000, Graham et al 2001a). …”

Evolutionary Analysis of the TPP-Dependent Enzyme Family

“…In addition to TPP, PO, ALS and GXC each require FAD (Bertagnolli and Hager 1993;Cromartie and Walsh 1976;Chang et al 1993;Koland JG 1982). Finally, SPDC and PPDC both consist only of PP-and Pyr-domains (Graupner et al 2000). In SPDC, the PPand Pyr-domains form the separate subunits (α-and β-) of an α 2 β 2 -heterotetramer.…”

The Phosphonopyruvate Decarboxylase from Bacteroides fragilis

“…The sulfopyruvate decarboxylase of the coenzyme M pathway, found in methaneforming bacteria (38), was identified as the closest homolog. Studies of the sulfopyruvate decarboxylase from Methanococcus jannaschii (39) have shown that the native enzyme is a dodecamer of 6 ␣-subunits (ComD 169 amino acids long; 34% sequence identity with the N-terminal half of the Ppyr decarboxylase) and 6 ␤-subunits (ComE 169 amino acids long; 39% sequence identity with the C-terminal half of Ppyr decarboxylase). It may be inferred that the ␣-and ␤-subunits of the sulfopyruvate decarboxylase correspond to N-terminal and Cterminal domains of the Ppyr decarboxylase.…”

“…Sulfopyruvate decarboxylase (39) and Ppyr decarboxylase are more distant members of a family of TPP-and Mg(II)-dependent decarboxylases that includes acetohydroxy acid synthase/acetolactate synthase (40), benzoylformate decar- boxylase (41), glyoxylate carboligase, indole pyruvate decarboxylase, pyruvate decarboxylase, the acetyl phosphate-producing pyruvate oxidase, and the acetate-producing pyruvate oxidase (42) (sequence identities between B. fragilis Ppyr decarboxylase and these proteins with a sequence range of 23-29%). The chemical reactions catalyzed by these enzymes are shown in Fig.…”