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Primary Hyperoxaluria and Kidney Stone Disease: Structural and Biochemical Analyses of Enzymes Involved in Glyoxylate Metabolism

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Primary Hyperoxaluria and Kidney Stone Disease: Structural and Biochemical Analyses of Enzymes Involved in Glyoxylate Metabolism
Murray, Michael S
Primary hyperoxaluria is characterized by elevated urinary oxalate output. The calcium salt of oxalate precipitates through the renal system as kidney stones. Primary hyperoxaluria type 1 (PH1) is caused by a defect or functional absence of the enzyme alanine:glyoxylate aminotransferase (AGT). Primary hyperoxaluria type 2 (PH2) is caused by mutations in the gene encoding glyoxylate reductase:hydroxy-pyruvate reductase (GRHPR). The structural and biochemical links between PH1-causing mutations in AGT and the disease phenotype have been well studied and characterized. However, there are few data on the normal activity of wild-type GRHPR or how the various PH2-causing mutations affect the enzyme. We have undertaken a detailed kinetic analysis of both wild-type GRHPR and three PH2-causing missense mutations under the hypothesis that this will lead to insight into abnormal glyoxylate metabolism leading to oxalate production. The results show that the mutations each affect different steps of the catalytic cycle. Additionally, we have solved the X-ray crystal structure of GRHPR in complex with an unknown molecule using a crystal contact mutation that leads to alternate packing within the crystal. Unlike previous structures, the new structure was solved in-house and did not require the time or expense required to utilize synchrotron radiation. Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most α-hydroxy acid oxidases, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur upon substrate binding. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with α-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen bonding network between Trp110, Leu191, Tyr134 and Tyr208. The loss of these interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. The data presented here support the hypothesis that the metabolic pathways of glyoxylate formation are tightly regulated. The intersection points of these pathways serve to control the flux of metabolites in healthy cells and are regulated by AGT, GRHPR, and GO. Knowledge of the pathways of oxalate formation and the molecular etiology of PH2 may lead to new methods of disease treatment for patients suffering from kidney stone disorders.
renal failure
Ross P. Holmes, Ph.D. (committee chair)
Roy R. Hantgan, Ph.D. (committee member)
Leslie B. Poole, Ph.D, (committee member)
Jacquelyn S. Fetrow, Ph.D. (committee member)
W. Todd Lowther, Ph.D. (committee member)
Murray, Michael S
2008-09-28T10:55:22Z (accessioned)
2010-06-18T18:58:06Z (accessioned)
null (available)
2008-09-28T10:55:22Z (available)
2010-06-18T18:58:06Z (available)
2008 (issued)
null (defenseDate)
Biochemistry & Molecular Biology (discipline)
Wake Forest University (grantor)
PHD (level)
http://hdl.handle.net/10339/14733 (uri)
etd-07212008-094507 (oldETDId)
Release the entire work immediately for access worldwide. (accessRights)
I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Wake Forest University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. (license)

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