Molybdenum cofactor (Moco) is a redox cofactor essential for bacterial growth under hypoxic and nutrient limiting environments, and therefore, is essential for persistence of pathogenic bacteria in mammalian hosts. Chronic bacterial infections are resistant to many antibiotics and cause the recurrence of acute symptoms. Although Moco biosynthesis has been shown to be essential for some pathogenic bacteria to cause chronic infections, the development of specific inhibitors has been hampered by a lack of understanding of the functions and mechanisms of the biosynthetic enzymes. The long-term goal of this project is to provide enzymological understanding of Moco biosynthesis in bacteria and its role in infectious disease. The current application focuses on the first committed step(s) of Moco biosynthesis where the characteristic pyranopterin structure of Moco is synthesized from guanine 5'-triphosphate (GTP). This transformation proceeds through an unprecedented mechanism in which the C-8 of GTP is inserted between the ribose C2' and C3'. While two enzymes (MoaA and MoaC) are known to be responsible for this transformation, their individual functions are currently under active debate. Recently, we reported the isolation of 3',8-cyclo- dihydro-GTP (3',8-cH2GTP) from in vitro MoaA assay solutions, and proposed that MoaA catalyzes the conversion of GTP to 3',8-cH2GTP, while MoaC catalyzes the conversion of 3',8-cH2GTP to cyclic pyranopterin monophosphate. In this application, we will test this hypothesis though three Specific Aims.
In Aim 1, the catalytic function and mechanism of MoaA will be investigated using the purification/derivatization-free 13C NMR method and substrate analogs.
In Aim 2, the structure-function relationship of MoaC will be investigated based on X-ray crystallography and in vivo and in vitro enzyme activity assays.
In Aim 3, putative MoaC reaction intermediates will be captured using MoaC active-site mutants or substrate analogs. The proposed research is significant because it will provide mechanistic insights into Moco backbone formation as well as the scientific basis for the future development of Moco biosynthesis inhibitors.

Public Health Relevance

The proposed research is relevant to public health because bacterial Moco biosynthesis is essential for pathogenic bacteria to persist in mammalian hosts, and its inhibition could eradicate chronic infections. Thus, the proposed research will contribute to developing fundamental knowledge that will help to combat persistent bacterial infectious diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM112838-02
Application #
9102114
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Gerratana, Barbara
Project Start
2015-07-01
Project End
2019-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Yokoyama, Kenichi; Lilla, Edward A (2018) C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products. Nat Prod Rep 35:660-694
Pang, Haoran; Yokoyama, Kenichi (2018) Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis. Methods Enzymol 606:485-522
Byer, Amanda S; Yang, Hao; McDaniel, Elizabeth C et al. (2018) Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate ? Is Central to Catalysis. J Am Chem Soc 140:8634-8638
Yokoyama, Kenichi (2018) Radical Breakthroughs in Natural Product and Cofactor Biosynthesis. Biochemistry 57:390-402
Yokoyama, Kenichi; Leimkühler, Silke (2015) The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria. Biochim Biophys Acta 1853:1335-49
Hover, Bradley M; Lilla, Edward A; Yokoyama, Kenichi (2015) Mechanistic Investigation of cPMP Synthase in Molybdenum Cofactor Biosynthesis Using an Uncleavable Substrate Analogue. Biochemistry 54:7229-36
Hover, Bradley M; Tonthat, Nam K; Schumacher, Maria A et al. (2015) Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis. Proc Natl Acad Sci U S A 112:6347-52
Hover, Bradley M; Yokoyama, Kenichi (2015) C-Terminal glycine-gated radical initiation by GTP 3',8-cyclase in the molybdenum cofactor biosynthesis. J Am Chem Soc 137:3352-9