Research is proposed in four specific enzyme mechanism problems where the enzymes catalyze metabolically important but chemically unobvious transformations. In each case design and testing of suicide substrates as mechanistic probes and potentially useful selective inhibitors are featured as part of the investigations. 1) Enzymic cleavage of 1-amino-1-carbosy-cyclopropane, a precursor of the fruit-ripening hormone ethylene, will be studied to analyze how the cyclopropane is activated for fragmentation in bacterial and plant metabolism. 2) The glutamine-dependent aromatizing aminations catalyzed by anthranilate synthetase and PABA-synthetase will be probed with pure enzymes and substrate analogs and reaction intermediates. 3) We will analyze pure hepatic cytochrome P450 isozymes for selectivity in processing and routes of suicidal autoinactivation with a) olefinic and acetylenic barbiturates, b) 17-d-ethynyl steroids c) the 7-thioacetyl steroid diuretic spironolactone. 4) The molecular mechanism of bacterial resistance to organomercurials involves the novel enzyme activity organomercury lyase. We plan to purify the enzyme and analyze the mechanism of cleavage of the cargon-mercury bond.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM020011-23
Application #
2173515
Study Section
Special Emphasis Panel (NSS)
Project Start
1987-09-30
Project End
1995-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
23
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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; 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
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

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