The basic pathway for the biosynthesis of inosine 5'-monophosphate (IMP) in vertebrates requires ten enzymatic conversions. Purine nucleotides are the building blocks of RNA and DNA and enzymes of the purine biosynthetic pathway are targets for anticancer, antiviral and antimicrobial drug discovery. Over the past decade three-dimensional structures for most of the enzymes were worked out, revealing many functional details. However, several unresolved issues remain. There are no structures for FGAM synthetase I (PurL, step 4), a 1400 amino acid protein with multiple domains and for FGAM synthetase II, an 800 amino acid protein requiring two additional gene products, PurS and PurQ. Likewise, there are no structures for FAICAR synthetase II (PurP, step 9) and IMP cyclohydrolase II (PurO, step 10) found in methanogenic archaebacteria. There are several different classes of AIR carboxylase (PurE, step 6) having different mechanisms, but only the structure of the class I enzyme is known. In addition, the proposed acylphosphate intermediates for the ATP-grasp enzymes (PurD, PurT, PurK, PurC and probably PurP) have never been characterized nor have the proposed iminophosphate intermediates for AIR synthetase (PurM, step 5) and PurL. No structures are available for the higher order complexes such as PurK/PurE, PurC/PurE, PurS/PurL/PurQ and PurD/PurM/PurN. The purpose of this proposal is to study the structure and function of purine biosynthetic enzymes and to provide structural information currently missing. The research will focus on enzyme mechanism, protein/protein interactions, substrate channeling and protein evolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073220-03
Application #
7175338
Study Section
Special Emphasis Panel (ZRG1-BPC-B (02))
Program Officer
Flicker, Paula F
Project Start
2005-02-01
Project End
2009-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
3
Fiscal Year
2007
Total Cost
$254,304
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Hicks, Katherine A; Ealick, Steven E (2016) Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway. Acta Crystallogr D Struct Biol 72:808-16
Fenwick, Michael K; Philmus, Benjamin; Begley, Tadhg P et al. (2016) Burkholderia glumae ToxA Is a Dual-Specificity Methyltransferase That Catalyzes the Last Two Steps of Toxoflavin Biosynthesis. Biochemistry 55:2748-59
Ye, Wenjie; Paul, Debamita; Gao, Lina et al. (2014) Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites. PLoS One 9:e115082
Hicks, Katherine A; O'Leary, Seán E; Begley, Tadhg P et al. (2013) Structural and mechanistic studies of HpxO, a novel flavin adenine dinucleotide-dependent urate oxidase from Klebsiella pneumoniae. Biochemistry 52:477-87
Sikowitz, Megan D; Cooper, Lisa E; Begley, Tadhg P et al. (2013) Reversal of the substrate specificity of CMP N-glycosidase to dCMP. Biochemistry 52:4037-47
Philmus, Benjamin; Abdelwahed, Sameh; Williams, Howard J et al. (2012) Identification of the product of toxoflavin lyase: degradation via a Baeyer-Villiger oxidation. J Am Chem Soc 134:5326-30
Huang, Siyu; Mahanta, Nilkamal; Begley, Tadhg P et al. (2012) Pseudouridine monophosphate glycosidase: a new glycosidase mechanism. Biochemistry 51:9245-55
Dessanti, Paola; Zhang, Yang; Allegrini, Simone et al. (2012) Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase. Acta Crystallogr D Biol Crystallogr 68:239-48
Fenwick, Michael K; Philmus, Benjamin; Begley, Tadhg P et al. (2011) Toxoflavin lyase requires a novel 1-His-2-carboxylate facial triad. Biochemistry 50:1091-100
French, Jarrod B; Neau, David B; Ealick, Steven E (2011) Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase. J Mol Biol 410:447-60

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