DIRECT SULFHYDRYLATION FOR METHIONINE BIOSYNTHESIS IN LEPTOSPIRA-MEYERI

A gene library of the Leptospira meyeri serovar semaranga strain Veldr at 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 ca rrying metY complemented Escherichia coli strains carrying mutations i h metB. The latter gene encodes cystathionine gamma-synthase, an enzym e which catalyzes the second step of the methionine biosynthetic pathw ay. The metY gene is 1,304 bp long and encodes a 443-amino-acid protei n with a molecular mass of 45 kDa as determined by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis. The deduced amino acid sequenc e of the Leptospira metY product has a high degree of similarity to th ose of O-acetylhomoserine sulfhydrylases from Aspergillus nidulans and Saccharomyces cerevisiae. A lower degree of sequence similarity was a lso found with bacterial cystathionine gamma-synthase. The L. meyeri m etY gene was overexpressed under the control of the T7 promoter. MetY exhibits an O-acetylhomoserine sulfhydrylase activity. Genetic, enzyma tic, and physiological studies reveal that the transsulfuration pathwa y via cystathionine does not exist in L. meyeri, in contrast to the si tuation found for fungi and some bacteria. Our results indicate, there fore, that the L. meyeri MetY enzyme is able to perform direct sulfhyd rylation for methionine biosynthesis by using O-acetylhomoserine as a substrate.