Nucleoside monophosphate synthesis provides the components of DNA, RNA, ATP and NAD and requires the action of phosphoribosyltransferases (PRTases) to transfer the ribose 5-phosphate group from its donor PRPP to specific heterocyclic bases. The PRTases are the site of several life-threatening hereditary disorders, and are legitimate targets for anti-parasitic, anti-microbial and anti-tumor agents. This consortium seeks to understand the structure and function of the PRTase enzyme group. Orotate PRTase, hypoxanthine-guanine PRTase and quinolinate PRTase have been crystallized and their three dimensional structures determined. The project seeks to reveal the structural and chemical details of enzymic catalysis through studies of OPRTase and QAPRTase. Using chemical quench techniques, the OPRTase kinetic mechanism will be detailed to permit understanding of newly discovered negative cooperativity of binding and catalysis. The OPRTase active site borders the subunit interface, and requires a mobile protein structural element (loop) contributed by the adjacent subunit. Structural interactions at the subunit interface may be responsible for cooperativity. The contribution to active site chemistry of the flexible element will be determined using point mutants and partial loop deletants and by determining the structure of enzyme complexed with novel synthetic bi-substrate analogue inhibitors. The catalytic function of a pair of carboxylate residues, common to Class 1 PRTases, will be determined. QAPRTase represents a newly discovered evolutionary origin and novel architecture (the Class 2 PRTase fold) for the PRTase enzyme group. The X-ray structure of QAPRTase will be refined and the catalytic role of active site residues structurally analogous to those in OPRTase will be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048623-06
Application #
2857168
Study Section
Biochemistry Study Section (BIO)
Project Start
1992-08-01
Project End
2001-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Temple University
Department
Miscellaneous
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Cook, Ian; Wang, Ting; Almo, Steven C et al. (2013) The gate that governs sulfotransferase selectivity. Biochemistry 52:415-24
Cook, Ian; Wang, Ting; Almo, Steven C et al. (2013) Testing the sulfotransferase molecular pore hypothesis. J Biol Chem 288:8619-26
Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A et al. (2012) Structure of Salmonella typhimurium OMP synthase in a complete substrate complex. Biochemistry 51:4397-405
Wang, Gary P; Hansen, Michael Riis; Grubmeyer, Charles (2012) Loop residues and catalysis in OMP synthase. Biochemistry 51:4406-15
Bello, Zainab; Stitt, Barbara; Grubmeyer, Charles (2010) Interactions at the 2 and 5 positions of 5-phosphoribosyl pyrophosphate are essential in Salmonella typhimurium quinolinate phosphoribosyltransferase. Biochemistry 49:1377-87
Bello, Zainab; Grubmeyer, Charles (2010) Roles for cationic residues at the quinolinic acid binding site of quinolinate phosphoribosyltransferase. Biochemistry 49:1388-95
Cao, Hong; Pietrak, Beth L; Grubmeyer, Charles (2002) Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase. Biochemistry 41:3520-8
Wang, G P; Cahill, S M; Liu, X et al. (1999) Motional dynamics of the catalytic loop in OMP synthase. Biochemistry 38:284-95
Wang, G P; Lundegaard, C; Jensen, K F et al. (1999) Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate. Biochemistry 38:275-83
Sharma, V; Grubmeyer, C; Sacchettini, J C (1998) Crystal structure of quinolinic acid phosphoribosyltransferase from Mmycobacterium tuberculosis: a potential TB drug target. Structure 6:1587-99

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