Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited

Abstract

The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysis demonstrated that metX is the second gene of an operon.The first step of the biosynthetic pathway leading to methionine is esterification of homoserine (6). However, the precursor differs according to the organism. In members of the family Enterobacteriaceae, O-succinyl-homoserine is formed by acylation of homoserine in the presence of O-succinyl-coenzyme A, catalyzed by homoserine O-succinyltransferase, the metA gene product (7,18). In gram-positive bacteria of the genus Bacillus and in fungi, esterification of homoserine occurs by an acetylation (3, 9, 13) catalyzed by a homoserine O-acetyltransferase.The genus Leptospira consists of two nomenspecies, Leptospira interrogans sensu lato (pathogenic) and Leptospira biflexa sensu lato (nonpathogenic), which belong to the order Spirochaetales (4). Previous studies indicated that Leptospira spp. synthesize most of their amino acids by the same biosynthetic pathways as those used by Escherichia coli (5).Our interest in methionine biosynthesis in Leptospira arose from the fact that radiotracer studies of biosynthetic pathways did not include any data for the methionine pathway (5). In this paper, we describe isolation and transcriptional organization of the metX gene, as well as the enzymatic characterization and regulation of its product, homoserine O-acetyltransferase.(This work represents a portion of a thesis submitted by P. Bourhy to the University of Paris VII, Paris, France, for the Ph.D.)Cloning of a DNA fragment from Leptospira meyeri which can complement an E. coli metA mutant. We had previously constructed a recombinant cosmid library and cloned the Leptospira metY gene, which complemented E. coli metB mutants (1). The size of the cloned DNA fragment (25 kbp) in the pb10 recombinant cosmid suggested that other methionine biosynthetic genes might be found on this fragment. In order to test this hypothesis, E. coli RC709 (metF63 pro-22; R. Clowes) and AB1932 (metA28 argH1 thi-1 lacY1 lacZ4 galK2 xyl-4 [or -5] tsx-6 F Ϫ ; E. A. Adelberg) were used for complementation experiments in supplemented M9 minimal medium (20) at 30°C. Both strains were electroporated with pb10. pb10 weakly complemented metA but not metF mutants. Further subcloning experiments allowed us to locate the Leptospira DNA able to complement E. coli metA and metB mutants more precisely within a 6.8-kbp fragment. Plasmid pb12 carrying a 6.8-kbp PstI fragment from pb10 in the pBR322 vector (22) still complemented E. coli metB and metA mutants (Fig.

DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

“…The 346 N-terminal residues had 24% identity and 40% similarity to the aminotransferase of Methanobacterium thermoautotrophicum (52). The 443 C-terminal residues had 41% identity and 56% similarity to the O-acetylhomoserine sulfhydrylase of Leptospira meyer (2). O-Acetylhomoserine sulfhydrylase synthesizes sulfur-containing amino acids from O-acetylhomoserine and sulfide.…”

Localization of the Leptospira interrogans metF gene on the CII secondary chromosome

“…The methionine genes are dispersed over the two chromosomes of Leptospira since metX and metY are located on CI [6,7] and metF on CII (see Section 3.1). However, the data concerning metX and metY had been performed with the L. meyeri strain Veldrat [7], which di¡ers from the L. interrogans Verdun strain used in this study. It was thus important to validate the ¢ndings for strain Verdun.…”

“…The metX and metY genes catalyze the ¢rst steps of the pathway [6,7] and the metF gene corresponds to the folate branch (this study). The methionine genes are dispersed over the two chromosomes of Leptospira since metX and metY are located on CI [6,7] and metF on CII (see Section 3.1).…”

“…The metX and metY genes catalyze the ¢rst steps of the pathway [6,7] and the metF gene corresponds to the folate branch (this study). The methionine genes are dispersed over the two chromosomes of Leptospira since metX and metY are located on CI [6,7] and metF on CII (see Section 3.1). However, the data concerning metX and metY had been performed with the L. meyeri strain Veldrat [7], which di¡ers from the L. interrogans Verdun strain used in this study.…”

“…The ¢rst step, esteri¢cation of homoserine, is catalyzed by a homoserine O-acetyltransferase (MetX) [7]. Then direct sulfhydrylation of O-acetylhomoserine to homocysteine involves MetY, an O-acetylhomoserine sulfhydrylase [6].…”