We intend to probe enzymatic reaction mechanisms by two main lines of approach, the first with mechanism-based enzyme inactivators, the second via study of fluorinated substrate analogs. Mechanism-Based Inactivators or Suicide Substrates require catalytic unmasking of a latent group by the enzyme at its active site, leading to covalent inactivation. The approach provides mechanistic information, structural information on active site catalytic components, and has real in vivo utility. We will investigate allylsulfoxide inactivators of amine oxidases, flavoprotein S-oxygenases, and live cytochrome P450 monooxygenases. We will also study the mechanism of action of neuroconvulsive allylglycine on glutamate decarboxylase, the lathyritic agent beta-aminopropionitrile on elastin lysyl oxidase, and fluorinated amino acid lactones on serine hydroxymethylase. Fluorinated substrates involve all four separate diastereomers of 2-fluorocitrate on various citrate-utilizing enzymes and also the preparation and utilization of chiral 2-(3H)-2-fluoroacetylCoA with claisen condensation enzymes (citrate synthase , malate synthase). Also 3-(3H)-3-fluoropyruvate will be prepared in chiral form and used with biotin-dependent carboxylases and thiamin-dependent decarboxylases to probe enzyme stereochemistry. In the fluorinated substrate work, enzyme stereospecificity, or lack thereof, is linked to molecular bases of toxicity.

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National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Massachusetts Institute of Technology
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Tsodikov, Oleg V; Hou, Caixia; Walsh, Christopher T et al. (2015) Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution. BMC Struct Biol 15:13
Walsh, Christopher T; Wencewicz, Timothy A (2014) Prospects for new antibiotics: a molecule-centered perspective. J Antibiot (Tokyo) 67:7-22
Setser, Jeremy W; Heemstra Jr, John R; Walsh, Christopher T et al. (2014) Crystallographic evidence of drastic conformational changes in the active site of a flavin-dependent N-hydroxylase. Biochemistry 53:6063-77
Walsh, Christopher T; O'Brien, Robert V; Khosla, Chaitan (2013) Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed Engl 52:7098-124
Parker, Jared B; Walsh, Christopher T (2013) Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin. Biochemistry 52:889-901
Malcolmson, Steven J; Young, Travis S; Ruby, J Graham et al. (2013) The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds. Proc Natl Acad Sci U S A 110:8483-8
Walsh, Christopher T; Wencewicz, Timothy A (2013) Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat Prod Rep 30:175-200
Walsh, Christopher T; Haynes, Stuart W; Ames, Brian D et al. (2013) Short pathways to complexity generation: fungal peptidyl alkaloid multicyclic scaffolds from anthranilate building blocks. ACS Chem Biol 8:1366-82
Haynes, Stuart W; Gao, Xue; Tang, Yi et al. (2013) Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin. ACS Chem Biol 8:741-8
Gao, Xue; Jiang, Wei; Jiménez-Osés, Gonzalo et al. (2013) An iterative, bimodular nonribosomal peptide synthetase that converts anthranilate and tryptophan into tetracyclic asperlicins. Chem Biol 20:870-8

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