Investigations of halogenated intermediates in the meta-fission pathway and of the tautomerase superfamily in biosynthetic pathways

Stack, Tyler Mason Maxwell
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The tautomerase superfamily (TSF) is a group of relatively short monomers (60-150 amino acid residues) that is characterized by a core β-α-β fold, along with an N-terminal proline. In the characterized TSF members, Pro-1 is a critical catalytic group. 4-oxalocrotonate tautomerase (4-OT) is the first characterized member of this superfamily, and is part of the bacterial meta-fission pathway in Pseudomonas putida mt-2. 5-Halo-2-hydroxy-2,4-pentadienoates are intermediates in a similar pathway in Comamonas testosteroni CNB-1, yet the intermediates contain a halogen. The enzymes in the Leptothrix cholodnii SP-6 meta-fission pathway are used as representative C. testosteroni enzymes. The presence of a halogen on these intermediates yielded two insights into how Nature processes halogenated species. First, 5-halo-2-hydroxy-2,4-pentadienoates are mechanism-based inactivators of 4-OT. Mass spectral and crystallographic analysis of the covalently inactivated enzymes showed distinct mechanisms for the fluoro- and bromo-/chloro-substituted species. A kinetic analysis suggests that the L. cholodnii 4-OT is more resistant to inactivation. Second, a stereochemical analysis on two vinylpyruvate hydratases (VPHs), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate, showed identical mechanisms. A stereochemical analysis in D₂O using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate showed that deuterons are incorporated stereospecifically and identically at the C-3 and C-5 positions of the products for both enzymes. These initial observations could suggest that the 4-OT-catalyzed reaction may have evolved in L. cholodnii (and C. testosteroni) to prevent inactivation, yet the VPH enzymes have similar mechanisms that could possibly be common to this group of enzymes. The common starting unit of streptogramin B and pyridomycin is 3-hydroxypicolinate (3-HP), which is likely derived from lysine. The biosynthetic steps required for the conversion of lysine to 3-HP have not been elucidated, but the genes for Pyr5 and SnbT have been identified. These two TSF members (72% identical, 87% similar) are implicated in the transformation. Preliminary work shows that Pyr5 and SnbT might function as tautomerases, due to the 1,3-keto-enol tautomerase activity with 2-hydroxypentadienoate and the inactivation by 2-oxo-3-pentynoate. The latter observation suggests a low pK [subscript a] for Pro-1, like that of 4-OT. A biosynthetic pathway consisting of four enzymes to convert lysine to 3HP is proposed.