Investigation of mosA, a protein implicated in rhizopine biosynthesis
Phenix, Christopher Peter
MosA is a protein found in Sinorhizobium meliloti L5-30 and has been suggested to be responsible for the biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine (3-MSI) from scyllo-inosamine (SI). However, we have shown MosA is a dihydrodipicolinate synthase (DHDPS) catalyzing the condensation of pyruvate with aspartate-β-semialdehyde (ASA). Since the DHDPS reaction occurs through a Schiff base aldol-type mechanism it was proposed that MosA could be an O-methyltransferase utilizing 2-oxo-butyrate (2-OB) as a novel methyl donor. This interesting yet unlikely possibility would explain MosA's role in the biosynthesis of 3-MSI without ignoring its similarity to DHDPS. Alternatively, MosA may have two catalytic domains one of which possesses a novel binding motif for S-Adenosyl methionine (SAM) to account for methyltransfer activity. In vitro demonstration of MosA’s methyltransferase activity is required to resolve this apparent contradiction.This dissertation describes the chemical synthesis of the rhizopines, investigation into whether MosA has a direct role in rhizopine biosynthesis and the thermodynamic characterization of compounds interacting with MosA as observed by isothermal titration calorimetry. Initial investigation into MosA’s methyltransferase activity began with 2-OB’s interaction with the enzyme. Inhibition experiments determined 2-OB is a competitive inhibitor with respect to pyruvate of the DHDPS reaction of MosA. Furthermore, protein mass spectrometry of MosA in the presence of 2-OB and sodium borohydride indicated that a Schiff base enzyme intermediate was indeed being formed providing evidence that the proposed mechanism may exist. However, neither of the rhizopines had any effect on the DHDPS activity and HPLC assays determined that no 3-MSI was being produced by MosA in the presence of SI and 2-OB. Furthermore, HPLC assays failed to detect methyl transfer activity by MosA utilizing the SAM as a methyl donor. Isothermal titration calorimetry provided thermodynamic characterization of the pyruvate and 2-OB Schiff base intermediates formed with MosA. In addition, ITC provided insight into the nature and thermodynamics of (S)-lysine’s inhibition of MosA. ITC failed to detect any interactions between the rhizopines or SAM with MosA. These results indicate that MosA is only a DHDPS and does not catalyze the formation of 3-MSI from SI as hypothesized in the literature.
DegreeDoctor of Philosophy (Ph.D.)
CommitteePaige, Matthew F.; Majewski, Marek; Delbaere, Louis T. J.; Verrall, Ronald E.
Copyright DateMay 2007
isothermal titration calorimetry