Corynebacterium glutamicumUtilizes both Transsulfuration and Direct Sulfhydrylation Pathways for Methionine Biosynthesis

Abstract

A direct sulfhydrylation pathway for methionine biosynthesis in Corynebacterium glutamicum was found. The pathway was catalyzed by metY encoding O-acetylhomoserine sulfhydrylase. The gene metY, located immediately upstream of metA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction of metY into C. glutamicum resulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase in O-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of the metB gene, which encodes cystathionine gamma-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or the metY gene was not enough to lead to methionine auxotrophy, an additional mutation in the metY or the metB gene resulted in methionine auxotrophy. The growth pattern of the metY mutant strain was identical to that of the metB mutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, an Escherichia coli metB mutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterial metY and metA genes. These data clearly show that C. glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. Although metY and metA are in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As with metA, methionine could also repress the expression of metY. The repression was also observed with metB, but the degree of repression was more severe with metY, which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway in C. glutamicum.

Bacterial methionine biosynthesis

“…1). However, studies on species including C. glutamicum (Hwang et al, 2002), B. subtilis (Auger et al, 2002) and P. putida (Vermeij & Kertesz, 1999) have demonstrated that the E. coli pathway is not ubiquitous. In this review, we have attempted to summarize the range of biochemical strategies that bacteria employ for synthesizing methionine.…”

“…Using cysteine as the source of the thiol group is more costly metabolically, but it is often more available than free sulfide, which is highly volatile. Examples of organisms in which both routes have been studied are C. glutamicum (Hwang et al, 2002) and Leptospira meyeri (Hwang et al, 2002;Picardeau et al, 2003), although in C. glutamicum the cystathionine b-lyase is encoded not by a metC gene, but by the more distant structural homologue, aecD (Ruckert et al, 2003).…”

Purification and Characterization ofO-Acetylserine Sulfhydrylase ofCorynebacterium glutamicum

“…12,13) But, there has been no report on the cysteine biosynthesis enzyme of C. glutamicum in purified form. To our knowledge, this is the first purification report of OASS of C. glutamicum.…”

Identification and Characterization ofglxR, a Gene Involved in Regulation of Glyoxylate Bypass inCorynebacterium glutamicum

“…Corynebacterial cell extracts were prepared as described previously (16). The enzymatic activities of ␤-galactosidase (25), malate synthase (8,15), isocitrate lyase (8,15), isocitrate dehydrogenase (13), and acetate kinase (41) were determined as described previously.…”