Direct Sulfhydrylation for Methionine Biosynthesis in Leptospira meyeri

The biosynthetic pathways of sulfur amino acids are well documented. Two alternative methionine biosynthetic pathways exist in microorganisms (Fig. ​(Fig.1).1). One, called the transsulfuration pathway, involves cystathionine formation, and the other bypasses cystathionine via direct sulfhydrylation of O-acylhomoserine to homocysteine (29). FIG. 1 Biosynthetic pathways of sulfur amino acids in E. coli (A) and S. cerevisiae (B). Enzyme steps: 1, O-succinylhomoserine transferase (metA); 1′, O-acetylhomoserine transferase; 2, cystathionine γ-synthase (metB); 3, cystathionine β-lyase ... In enteric bacteria, the sulfur atom is incorporated first into a serine ester (O-acetylserine) to yield cysteine (16). Sulfur is then transferred from cysteine to homocysteine via transsulfuration. In Escherichia coli, it requires the sequential action of cystathionine γ-synthase (EC 4.2.99.9), the product of the metB gene (7), and cystathionine β-lyase (EC 4.4.1.8), the metC gene product (1), with the intermediary formation of cystathionine (Fig. ​(Fig.1A,1A, steps 2 and 3). The direct sulfhydrylation pathway has been reported to be the main pathway for homocysteine biosynthesis in Saccharomyces cerevisiae (5) and bacteria such as Brevibacterium flavum and Pseudomonas aeruginosa (10, 23). In S. cerevisiae, which is the best-studied example, the direct synthesis of homocysteine is catalyzed by an O-acetylhomoserine sulfhydrylase, the Met25 (or Met17) product (Fig. ​(Fig.1B,1B, step 4) (5, 34). The resulting homocysteine is used as a direct precursor for methionine and is converted to cysteine via the reverse transsulfuration pathway (Fig. ​(Fig.1B,1B, steps 5 and 6). In addition, it should be kept in mind that the ester of homoserine used for homocysteine biosynthesis differs depending on the organisms: enteric bacteria use O-succinylhomoserine, while fungi and most gram-positive bacteria use O-acetylhomoserine (Fig. ​(Fig.1,1, steps 1 and 1′) (for a review, see reference 33). Little is presently known about the regulation of the metabolite flux of the methionine pathway. However, it has been reported that the control at the enzymatic level in bacteria and S. cerevisiae occurred at an early step of the methionine biosynthetic pathway. The first enzyme of the methionine biosynthetic pathway in E. coli, O-succinylhomoserine transferase, is feedback inhibited by methionine and S-adenosylmethionine (20), while the activity of O-acetylhomoserine transferase from S. cerevisiae is inhibited only by S-adenosylmethionine (6). In previous work, we demonstrated that O-acetylhomoserine transferase activity in Leptospira meyeri is not regulated by methionine and/or S-adenosylmethionine (2). Our goal was to investigate the evolution of sulfur metabolism in L. meyeri. We report here the construction of a representative cosmid L. meyeri DNA library and the cloning of a biosynthetic gene, metY, which complements E. coli metB mutants. Analysis of the inferred L. meyeri MetY amino acid sequence, growth impairment of E. coli mutants carrying metY, and results of enzymatic assays allow us to propose a direct sulfhydrylation pathway catalyzed by an O-acetylhomoserine sulfhydrylase.