Direct sulfhydrylation for methionine biosynthesis in Leptospira meyeri.
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 metY complemented Escherichia coli strains carrying mutations in metB. The latter gene encodes cystathionine gamma-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 the Leptospira metY product has a high degree of similarity to those of O-acetylhomoserine sulfhydrylases from Aspergillus nidulans and Saccharomyces cerevisiae. A lower degree of sequence similarity was also found with bacterial cystathionine gamma-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.
Belfaiza
J
J
Faculté des Sciences d'El-Jadida, Université Chouaib Doukkali, El-Jadida, Morocco.
Martel
A
A
Margarita
D
D
Saint Girons
I
I
D000595:Amino Acid Sequence; D019757:Carbon-Oxygen Lyases / Q000235:genetics; D003001:Cloning, Molecular; D003547:Cysteine Synthase; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007919:Leptospira / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D008715:Methionine; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases