Studies on the Biosynthesis of Cysteine in Mycobacterium tuberculosis and Studies on the Biosynthesis of Vitamin B6 in Bacillus subtilis
|Cysteine is one of the 22 natural amino acids used to make proteins. Three pathways are currently known for the biosynthesis of cysteine and all include sulfide as the sulfur source. A new pathway for cysteine biosynthesis was elucidated and reconstituted from Mycobacterium tuberculosis. This pathway involves a protein bound thiocarboxylate (CysO-SH) as the sulfide donor, similar to thiamin and molybdopterin biosynthesis. MoeZ, a paralog of ThiF (thiamin) and MoeB (molybdopterin), transfers sulfide onto CysO from an unidentified source. Cysteine synthase M (CysM) catalyzes the addition of O-acetylserine to the carboxy terminus of the protein bound thiocarboxylate to generate a CysO-cysteine adduct. A protease, Mec+, hydrolyzes the CysO-cysteine adduct to release cysteine and regenerate CysO. Mec+ contains the JAMM motif and this work provides the first functional characterization of the JAMM motif in prokaryotes. This pathway could be important for M. tuberculosis under conditions of oxidative stress, as it would provide a more stable source of sulfide than the traditional pathways for cysteine formation. Vitamin B6, an essential cellular cofactor, is biosynthesized by bacteria and lower eukaryotes and is required in the human diet. Two de novo pathways for PLP biosynthesis were known, however, the most common pathway involving the YaaD and YaaE family of genes had not been reconstituted. The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) were identified as ribose-5-phosphate, glyceraldehyde-3-phosphate and glutamine; the product is pyridoxal-5?-phosphate. In addition to PLP formation, we identified three activities catalyzed by the YaaD subunit of PLP synthase, including ribosephosphate isomerase activity, triose phosphate isomerase activity, and adduct formation with ribulose-5-phosphate. We also investigated the early steps of the mechanism of pyridoxal-5?-phosphate formation. In the absence of triose, PLP synthase forms a stable intermediate which absorbs at 320nm. This intermediate then reacts with glyceraldehyde-3-phosphate to form PLP.
|Cysteine biosynthesis in Mycobacterium tuberculosis
|Vitamin B6 biosynthesis
|Studies on the Biosynthesis of Cysteine in Mycobacterium tuberculosis and Studies on the Biosynthesis of Vitamin B6 in Bacillus subtilis
|dissertation or thesis