X-Ray Crystal Structures Of Enzymes In Purine/Pyrimidine Salvage Pathway, Sulfur Assimilation Pathway And Structure-Based Design And Organic Synthesis Of A Lightactivated Inhibitor For Thiamin Biosynthetic Enzymes

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The structure of Streptococcus pyogenes uridine phosphorylase (UP) in the salvage pathway reveals that it uses different residues to stabilize the transition state. Sequence alignment of the enzyme uncovers a subclass of uridine phosphorylases that contain these active site residues. These data, together with the specificity loop of uridine phosphorylases, may be used to distinguish between UP and purine nucleoside phosphorylase (PNP) enzymes at the level of primary sequence, and thus may allow proper annotation. In Bos taurus, the active site of its purine nucleoside phosphorylase can accommodate the major by-product of DNA damage, pyrimidopurinone 2'-deoxyribose, and cleave its glycosidic bond. The base-binding site of the enzyme helps explain why the reaction cannot proceed in the reverse direction. The structure of a protein complex, ThiS-ThiG, in the thiazole biosynthetic pathway was used to rationally design and synthesize a light-activated analog. Preliminary characterization of the analog demonstrates that it could be covalently linked to ThiS using intein chemistry. We anticipate that this analog will be of use for the structural and mechanistic characterization of thiazole synthase. The crystallization and diffraction screenings of selected enzymes in the methionine biosynthetic and sulfur assimilation pathways of Wolinella succinogenes were carried out. The crystallization conditions for the MetY/homocysteine complex have been optimized from 8 Angstrom to 4 Angstrom resolution by screening with different cryoreagents. Detergent screens for the apo-MetY generated small crystals, which were used for X-ray diffraction experiments. The MetY structure, which is the first o-acetylhomoserine sulfhydrylase, has been determined. A search for structurally homologous proteins reveals that MetY has the same fold as cystathionine gamma lyase and methionine gamma lyase. The active sites of these enzymes, which contain PLP, share a high degree of structural similarity, suggesting that MetY belongs to the [gamma]-elimination subclass of Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides useful insight to the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate.

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