Nature assembles thousands of polyketide (PK) and nonribosomal peptide (NRP) natural product antibiotics on multimodular enzymatic assembly lines. Growing chains are tethered as a series of elongating covalent acyl-S-protein intermediates, attached by phosphopantetheinyl chains to carrier protein domains, typically one per module. This proposal examines catalytic machinery and chemical logic for two sets of maturation steps in antibiotic natural products. Nascent scaffolds are subject to tailoring reactions by dedicated enzymes that often are crucial for maturation of target biological activities.
In Specific Aim 1 we prose examination of scope and mechanism of two types of oxidative tailoring enzymes for NRP and NR-PK hybrids: halogenases and oxygenases. Tailoring halogenases use either FADH2 or mononuclear nonheme FeII, dependent on electronic demand of the carbon site in the antibiotic. We plan to use halogenase gene probes to clone biosynthetic gene clusters, e.g. for kutzneride, and examine scope of the halogenative tailoring. We shall also examine the activity and mechanism of tailoring oxygenases that act in antibiotic scaffold maturations.
Specific Aim 2 addresses catalytic domains in assembly lines involved in novel chemistry during chain elongation and termination steps. These include ester backbone linkages in place of amides, the recruitment of transglutaminase homologs as condensation domains that act in trans, formation of C-terminal pyrrolidinedione rings, and reductive chain termination steps.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM020011-38
Application #
7546565
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Jones, Warren
Project Start
1987-09-30
Project End
2011-11-30
Budget Start
2008-12-01
Budget End
2009-11-30
Support Year
38
Fiscal Year
2009
Total Cost
$658,648
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
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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