Exploring Novel Enzymology In Bacterial Metabolism: Cysteine Synthase, Urate Oxidase, And Bacimethrin Biosynthesis.

Abstract

The practice of biological chemistry in the post-genomic era has included increasingly broad and detailed interrogation of cellular processes at the molecular level. In particular, enzymology has been equipped with a wide range of physical and analytical tools to study in detail chemical reactions catalyzed by enzymes. The present work describes studies aimed at characterizing enzymes involved in bacterial metabolism. First, a pre-steady-state kinetic analysis is described of CysM, a cysteine synthase from Mycobacterium tuberculosis. This analysis led to two principal results. O-phospho-L-serine was identified as the immediate biosynthetic precursor for cysteine in its CysM-mediated biosynthesis in M. tuberculosis. The substrate for CysM and related enzymes in bacteria was previously assumed to be O-acetyl-Lserine. The study also resulted in the first detailed pre-steady-state kinetic characterization of sulfur transfer from a small sulfur carrier protein (CysO) to an enzyme-bound intermediate in a biosynthetic pathway. The second study involved biochemical characterization of the HpxO enzyme from Klebsiella pneumoniae. This enzyme catalyzes the oxidation of uric acid to 5hydroxyisourate as part of the purine catabolic pathway. The activity of HpxO was shown to depend on flavin adenine dinucleotide (FAD), in contrast to all previouslystudied urate oxidase enzymes, which employ a cofactor-independent chemical mechanism. The results confirmed the existence of a novel mechanistic paradigm in purine catabolism. A series of HpxO active-site mutants were generated and characterized kinetically in order to gain further insight into the HpxO-catalyzed mechanism of urate hydroxylation, in the context of its X-ray crystal structure. The third study reported here involved characterization of a biosynthetic pathway for bacimethrin, a thiamin antimetabolite. We identified a previously unknown biosynthetic pathway for bacimethrin in Clostridium botulinum and reconstituted in vitro the three enzymatic activities which are responsible for conversion of cytidine monophosphate to bacimethrin. We also investigated the activity of a thiaminase I enzyme found in the C. botulinum bacimethrin biosynthetic cluster. Our results implicate thiaminase I as a potentiator of bacimethrin toxicity and indicate a possible role for this enzyme in extracellular salvage of thiazole for thiamin biosynthesis.