Direct Sulfhydrylation for Methionine Biosynthesis in Leptospira meyeri

Abstract

A gene library of the Leptospira meyeri serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a Leptospira DNA fragment carrying metYcomplemented Escherichia coli strains carrying mutations inmetB. The latter gene encodes cystathionine γ-synthase, an enzyme which catalyzes the second step of the methionine biosynthetic pathway. The metY gene is 1,304 bp long and encodes a 443-amino-acid protein with a molecular mass of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of theLeptospira metY product has a high degree of similarity to those of O-acetylhomoserine sulfhydrylases fromAspergillus nidulans and Saccharomyces cerevisiae. A lower degree of sequence similarity was also found with bacterial cystathionine γ-synthase. The L. meyeri metY gene was overexpressed under the control of the T7 promoter. MetY exhibits an O-acetylhomoserine sulfhydrylase activity. Genetic, enzymatic, and physiological studies reveal that the transsulfuration pathway via cystathionine does not exist in L. meyeri, in contrast to the situation found for fungi and some bacteria. Our results indicate, therefore, that the L. meyeri MetY enzyme is able to perform direct sulfhydrylation for methionine biosynthesis by using O-acetylhomoserine as a substrate.

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

“…Three genes have been identi¢ed for the leptospiral methionine biosynthetic pathway. 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).…”

“…A cosmid library of L. interrogans serovar icterohaemorraghiae strain Verdun was made as described for another Leptospira species [6]. A recombinant cosmid (XG10) harboring an insert of around 40 kb in size was cleaved with the Sau3A restriction endonuclease according to the manufacturer's speci¢cations.…”

“…The [K-33 P]dATP labeling of the probes was performed with the Megaprime kit (Amersham). Several probes originating from L. interrogans strain Verdun were used: (i) plasmid pAIL4 was used as a probe for the asd gene [12]; (ii) a 530-bp probe speci¢c for the metY gene [6] was synthesized with two oligonucleotides 5P-CAAGAAATTGTTGCGTCTTC-3P and 5P-CTAGC-GCTTCTGGAAC-3P by polymerase chain reaction ; (iii) plasmid pGMetF3 was used as a probe for the metF gene. Hybridizations were carried out for 18 h at 65³C.…”

“…Molecular and biochemical data have shown that the ¢rst part of the methionine pathway for Leptospira, a bacterium belonging to the spirochete phylum, is similar to that from yeast [6]. The ¢rst step, esteri¢cation of ho-moserine, is catalyzed by a homoserine O-acetyltransferase (MetX) [7].…”

“…The ¢rst step, esteri¢cation of ho-moserine, is catalyzed by a homoserine O-acetyltransferase (MetX) [7]. Then direct sulfhydrylation of O-acetylhomoserine to homocysteine involves MetY, an O-acetylhomoserine sulfhydrylase [6]. The metX and metY genes, organized in an operon, belong to the large CI chromosome of Leptospira.…”