Complex enzymes perform multi-step catalysis in biosynthetic pathways in which labile or insoluble intermediates are channeled or transported between active sites. We propose to continue our structural studies of one complex enzyme system, the glutamine amidotransferases, and to initiate work on a second, the curacin polyketide synthase. The glutamine amidotransferases are biosynthetic enzymes that generate ammonia from glutamine in one active site and channel it to a second active site for addition to a second substrate.
In Aim 1, we will continue studies of GatCAB, which catalyzes the second half of tRNAGln or tRNAAsn aminoacylation in organisms lacking a glutamine tRNA synthetase or asparagine tRNA synthetase. Specifically, we will examine complexes of Aquifex aeolicus GatCAB with tRNA and substrate-like small molecules. These studies aim to understand how the GatB subunit recognizes mis-charged Glu-tRNAGln or Asp-tRNAAsn, how tRNA binding stimulates ammonia production in the GatA subunit, how ammonia is channeled between subunits, and what is the structure-based mechanism of nucleophilic addition of nitrogen in GatB to produce Gln-tRNAGln or Asn-tRNAAsn.
In Aims 2 and 3, we will focus on a new class of complex enzymes, the modular polyketide synthases (PKSs). Microbes produce an astounding variety of polyketide natural products, which are an important source of new bio-active molecules that can be exploited as lead compounds for development of new pharmaceuticals. In these mega-enzymes, a carrier domain transports insoluble intermediates between enzyme active sites. Our subject is the PKS for curacin A, a polyketide with anti-tubulin activity. We will study the structural biochemistry of three unusual chemical features of curacin: a cyclopropane ring, a cis double bond, and a terminal alkene. We will produce relevant catalytic domains encoded by the 75-kilobase curacin gene cluster, determine crystal structures of the domains and of their substrate or analog complexes, and examine catalytic specificity. The result of these studies will be an understanding of how the curacin PKS carries out its unusual chemical transformations, and to what extent the catalytic domains can tolerate substrate variants.

Public Health Relevance

Many enzymes perform complex multi-step chemical reactions. This project will examine the details of substrate interactions with several complex enzymes by solving crystal structures of enzyme-substrate or -analog complexes. The goal is to understand how catalytic domains of proteins channel a reaction product to the site of the next reaction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042303-22
Application #
8097928
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Sechi, Salvatore
Project Start
1990-02-01
Project End
2012-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
22
Fiscal Year
2011
Total Cost
$366,703
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Maloney, Finn P; Gerwick, Lena; Gerwick, William H et al. (2016) Anatomy of the ?-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate. Proc Natl Acad Sci U S A 113:10316-21
Skiba, Meredith A; Sikkema, Andrew P; Fiers, William D et al. (2016) Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase. ACS Chem Biol 11:3319-3327
Smith, Amber Marie; Brown, William Clay; Harms, Etti et al. (2015) Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase. J Biol Chem 290:5226-39
Khare, Dheeraj; Hale, Wendi A; Tripathi, Ashootosh et al. (2015) Structural Basis for Cyclopropanation by a Unique Enoyl-Acyl Carrier Protein Reductase. Structure 23:2213-23
Bernard, Steffen M; Akey, David L; Tripathi, Ashootosh et al. (2015) Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases. ACS Chem Biol 10:1340-51
Li, Yang; Dodge, Greg J; Fiers, William D et al. (2015) Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase. J Am Chem Soc 137:7003-6
Fiers, William D; Dodge, Greg J; Li, Yang et al. (2015) Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of ?-processing domains via diffusible, synthetic substrates. Chem Sci 6:5027-5033
Smith, Janet L; Skiniotis, Georgios; Sherman, David H (2015) Architecture of the polyketide synthase module: surprises from electron cryo-microscopy. Curr Opin Struct Biol 31:9-19

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