The molybdenum cofactor (Moco) is present in four different enzyme families which catalyze important transformations in the global carbon, sulfur and nitrogen cycles. Moco consists of a mononuclear molybdenum coordinated to the sulfur atoms of a dithiolene group which is part of a substituted pterin derivative termed molybdopterin. Enzymes containing this cofactor are found in all three phylogenetic kingdoms of life, and biosynthesis of the cofactor follows the same pathway, both in prokaryotes and in eukaryotes including plants and human. Moco deficiency is inherited as an autosomal recessive trait and is a severe disease in humans which manifests itself in neurological abnormalities leading to premature death in the affected individuals. The experiments outlined in this proposal address two important aspects of Moco, namely its biosynthesis and its detailed function once it has been incorporated into the target enzymes.
The specific aims presented in this proposal therefore are: (i) Characterization of the early steps in Moco biosynthesis through structural studies on the enzyme MoaC. MoaC most likely either catalyzes the first step in Moco biosynthesis starting with a phosphorylated guanosine, or the second step in which A molybdopterin precursor is generated which still lacks the sulfur atoms. (ii) Crystal structure analysis of molybdopterin synthase which incorporates the sulfur atoms into a molybdopterin precursor generating molybdopterin. The small subunit of the heterodimeric molybdopterin synthase contains an unusual C-terminal thiocarboxylate which serves as the sulfur donor during catalysis. (iii) Crystal structure determination of MogA which might function as a molybdochelatase and thus would be responsible for incorporating molybdenum, which is present in the cell as molybdate, into the molybdopterin. (iv) High resolution structure analysis of the Moco-containing enzyme dimethylsulfoxide reductase including characterization of different oxidation states and complexes of the enzyme with substrates and inhibitors which will lead to a detailed characterization of the catalytic mechanism. (v) Crystal structure analysis of biotin sulfoxide reductase, which is related in sequence to dimethylsulfoxide reductase but differs in substrate specificity. Comparative studies of the two enzymes will lead to a more general understanding of the roles Moco is playing in enzymes containing this cofactor.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054835-02
Application #
6150652
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Laughlin, Maren R
Project Start
1999-03-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$169,787
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Luther, Kelvin B; Schindelin, Hermann; Haltiwanger, Robert S (2009) Structural and mechanistic insights into lunatic fringe from a kinetic analysis of enzyme mutants. J Biol Chem 284:3294-305
Zhao, Gang; Li, Guangtao; Schindelin, Hermann et al. (2009) An Armadillo motif in Ufd3 interacts with Cdc48 and is involved in ubiquitin homeostasis and protein degradation. Proc Natl Acad Sci U S A 106:16197-202
Zhao, Gang; Li, Guangtao; Zhou, Xiaoke et al. (2009) Structural and mutational studies on the importance of oligosaccharide binding for the activity of yeast PNGase. Glycobiology 19:118-25
Lees, Nicholas S; Hänzelmann, Petra; Hernandez, Heather L et al. (2009) ENDOR spectroscopy shows that guanine N1 binds to [4Fe-4S] cluster II of the S-adenosylmethionine-dependent enzyme MoaA: mechanistic implications. J Am Chem Soc 131:9184-5
Schmitz, Jennifer; Chowdhury, Mita Mullick; Hanzelmann, Petra et al. (2008) The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins. Biochemistry 47:6479-89
Lee, Imsang; Schindelin, Hermann (2008) Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell 134:268-78
Li, Guangtao; Zhao, Gang; Schindelin, Hermann et al. (2008) Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD. Biochem Biophys Res Commun 375:247-51
Daniels, Juma N; Wuebbens, Margot M; Rajagopalan, K V et al. (2008) Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency. Biochemistry 47:615-26
Tian, Geng; Kober, Franz-Xaver; Lewandrowski, Urs et al. (2008) The catalytic activity of protein-disulfide isomerase requires a conformationally flexible molecule. J Biol Chem 283:33630-40
Zhao, Gang; Zhou, Xiaoke; Wang, Liqun et al. (2007) Studies on peptide:N-glycanase-p97 interaction suggest that p97 phosphorylation modulates endoplasmic reticulum-associated degradation. Proc Natl Acad Sci U S A 104:8785-90

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