Non-ribosomal peptide synthetases (NRPSs) produce peptides with antibiotic and anticancer activities and are therefore a target for combinatorial or genetic engineering to create catalysts that could generate novel peptides. NRPSs are modular proteins that contain multiple catalytic domains expressed as a single polypeptide. During synthesis, the nascent peptide is transferred from one catalytic domain to the next for further elongation or chemical modification. We have determined the X-ray crystal structures of two adenylate-forming enzymes that suggest that, at different steps of the reaction, the orientation of the C-terminal domain differs by 150 degrees. We have proposed that the closely-related NRPS adenylation domains, which activate the amino acid building blocks and covalently attach them to a second NRPS carrier protein domain, also adopt these two conformations. The magnitude of, and the manner in which these enzymes use, this change is striking and suggests that efforts to engineer the NRPS enzymes to make novel pharmaceuticals will require that steps are taken to avoid steric clashes that arise from the rotation of downstream domains. This domain alternation hypothesis will be investigated through x-ray crystallographic and biochemical analyses of three adenylate-forming enzymes, including a three-domain NRPS. Specifically, we will determine the structures a) acetyl-CoA synthetase, b) an aryl-CoA synthetase, c) a three-domain NRPS protein of which we have expressed a truncated two-domain adenylation domain-carrier protein domain fragment in an active form, and d) a two-domain NRPS protein, which we have crystallized, that serves as the amino acid acceptor for the adenylation domain. Through our structural work and biochemical analyses we will gain insight into the catalytic mechanism of these important NRPS domains, providing the structural foundation for efforts to engineer these catalysts for the development of new drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068440-04
Application #
7250250
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Flicker, Paula F
Project Start
2004-07-01
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
4
Fiscal Year
2007
Total Cost
$295,359
Indirect Cost
Name
Hauptman-Woodward Medical Research Institute
Department
Type
DUNS #
074025479
City
Buffalo
State
NY
Country
United States
Zip Code
14203
Miller, Bradley R; Gulick, Andrew M (2016) Structural Biology of Nonribosomal Peptide Synthetases. Methods Mol Biol 1401:3-29
Drake, Eric J; Miller, Bradley R; Shi, Ce et al. (2016) Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529:235-8
Kholodar, Svetlana A; Allen, C Leigh; Gulick, Andrew M et al. (2015) The role of phosphate in a multistep enzymatic reaction: reactions of the substrate and intermediate in pieces. J Am Chem Soc 137:2748-56
Mitchell, Carter A; Tucker, Alex C; Escalante-Semerena, Jorge C et al. (2015) The structure of S. lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain. Proteins 83:575-81
Miller, Bradley R; Sundlov, Jesse A; Drake, Eric J et al. (2014) Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases. Proteins 82:2691-702
Russo, Thomas A; Olson, Ruth; Macdonald, Ulrike et al. (2014) Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect Immun 82:2356-67
Allen, C Leigh; Gulick, Andrew M (2014) Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein. Acta Crystallogr D Biol Crystallogr 70:1718-25
Kholodar, Svetlana A; Tombline, Gregory; Liu, Juan et al. (2014) Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin. Biochemistry 53:3423-31
Wurst, Jacqueline M; Drake, Eric J; Theriault, Jimmy R et al. (2014) Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine. ACS Chem Biol 9:1536-44
Neres, João; Engelhart, Curtis A; Drake, Eric J et al. (2013) Non-nucleoside inhibitors of BasE, an adenylating enzyme in the siderophore biosynthetic pathway of the opportunistic pathogen Acinetobacter baumannii. J Med Chem 56:2385-405

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