Revealing O-acetylhomoserine sulfhydrylase involved in direct sulfhydrylation pathway in Clostridium tetani

Bacterial methionine biosynthesis is an attractive target for research due to its central role in cellular metabolism, as most steps of this pathway are missing in mammals. Up to now little is known about sulfur metabolism in pathogenic Clostridia species, making the study of the enzymes of Cys/Met metabolism in Clostridium tetani particularly relevant. Analysis of the C. tetani genome has shown that the bacterium is capable of synthesizing methionine by direct sulfhydration. In this study, we describe purification of recombinant O-acetylhomoserine sulfhydrylase, a member of the Cys/Met metabolism pyridoxal 5'-phosphate-dependent enzyme family, from C. tetani for the first time. The gene encoding O-acetylhomoserine sulfhydrylase was cloned into the pET-28a(+) vector and expressed in Escherichia coli. The expression product was purified and identified as a 462-amino acid protein with a molecular mass of ∼50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the C. tetani enzyme showed a high degree of similarity to O-acetylhomoserine sulfhydrylases from other bacterial sources. We confirmed the O-acetylhomoserine sulfhydrylase activity, and found the enzyme to be optimally active at pH 7.5 and 50 °C. The native enzyme assembles into a homotetramer of approx. 200 kDa as revealed by gel filtration. The obtained enzyme is capable of l-methionine formation using methanethiol as a sulfur source, that has been revealed by ¹H NMR spectral data. These findings broaden the understanding of the role of O-acetylhomoserine sulfhydrylase in C. tetani Cys/Met metabolism and provide a basis for its future investigations and research.