Molybdenum cofactor (Moco) is a redox cofactor found in almost all organisms. In humans, it is essential for normal brain development, and mutations in Moco biosynthetic genes cause the fatal and currently incurable disease, Moco deficiency (MoCD). In pathogenic bacteria, Moco is essential for their growth under hypoxic and nutrient limiting environments, and therefore essential for pathogen persistence in mammalian hosts. Chronic bacterial infections are resistant to many antibiotics and cause the recurrence of acute symptoms. However, the development of therapeutics against MoCD or antibiotics targeting Moco biosynthesis in pathogenic bacteria are currently difficult due to our limited understanding of the mechanism of Moco biosynthesis. The long-term goal of this project is to provide a mechanistic understanding of Moco biosynthesis in pathogenic bacteria as well as in humans. The focus of the current application is the mechanism of two enzymes (MoaA and MoaC) responsible for the formation of the pyranopterin structure of Moco from guanine 5'-triphosphate (GTP). While the catalytic functions of MoaA and MoaC had remained ambiguous for >20 years, we recently demonstrated that MoaA catalyzes the transformation of GTP into 3',8-cyclo-dihydro-GTP (3',8-cH2GTP), while MoaC catalyzes the conversion of 3',8-cH2GTP to cPMP. In this application, we will investigate the catalytic mechanisms of MoaA and MoaC in both humans and bacteria through the following three Aims.
In Aim 1, the redox function of 4Fe-4S clusters in MoaA will be investigated both in the resting state and during turnover to address one of the key unsolved mysteries of the radical SAM enzyme mechanisms.
In Aim 2, the function of the C-terminal tail of MoaA and the mechanism of peptide rescue of MoCD-causing mutations will be investigated through NMR, X-ray crystallography and biochemical assays using bacterial and human enzymes.
In Aim 3, we will test a covalent and non-covalent mechanisms for MoaC catalysis and investigate the generality of mechanism-based inhibition of bacterial and human enzymes. The proposed research is significant because it will provide mechanistic insights into the formation of the Moco backbone and the scientific basis for future development of Moco biosynthesis inhibitors and novel therapeutics to treat MoCD.
The proposed research is relevant to public health because bacterial Molybdopterin Cofactor (Moco) biosynthesis is essential for pathogenic bacteria to persist in mammalian hosts, and its inhibition could eradicate chronic infections. Additionally, in humans, Moco biosynthesis is essential for proper brain development, and mutations in human Moco biosynthetic genes causes the generally fatal disease, Moco deficiency. Thus, the proposed research will contribute to developing fundamental knowledge that will help combat both bacterial infectious diseases as well as a hereditary human disease.
|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|