Protein thiocarboxylates are sulfur carrier proteins involved in some important pathways such as thiamine, cysteine and molybdopterin biosynthetic pathways. They have some common features: 1) they're usually small proteins less than 10 kDa; 2) the C-terminus is floppy and contains Gly-Gly sequence; 3) the formation of thiocarboxylate takes two steps including ATP activation to form protein-AMP and sulfur incorporation to form protein thiocarboxylate. Though protein thiocarboxylates have been thoroughly studied and a fluorescein-labeling method has been developed to detecting these proteins, there lacks a method in efficiently enriching and identifying them. Hence, in this research, a biotin-sulfonazide molecule was designed for labeling and identifying thiocarboxylate group through thioacid-azide reaction. Using this method, a ThiS/MoaD family protein was detected in S. coelicolor. By enriching with streptavidin resin and digesting with trypsin, this ThiS/MoaD family protein was successfully identified as SCO4294 in S. coelicolor and differentiated by its characteristic glycine-glycine C-terminal. This thiS-like protein (SCO4294) in S. coelicolor was found to locate conservatively beside a protein named threonine synthase (SCO4293). Therefore, both the two proteins were cloned and overexpressed in E. coli. The purified proteins were put into reaction with O-phospho-L-homoserine as the substrate. The reaction products were found to be homocysteine (major product), threonine and homoserine. Vinylglycine was also observed if the substrate is in much excess than thiocarboxylate protein. It is proposed that the production of homocysteine is a process involved in methionine biosynthetic pathway.

Identification and characterization of new protein thiocarboxylate in bacteria

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.