Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human pathological conditions associated with the accumulation of this compound in the blood and/or urine. This proposal outlines the continuation of integrated experimental and computational studies aimed at understanding the fundamental biochemistry and regulation of oxalate decarboxylase (OxDC), an enzyme that catalyzes the conversion of oxalate to carbon dioxide and formate. Both of these products are non-toxic and so OxDC has the potential for clinical use in treating urolithiasis and/or preventing the formation of calcium oxalate-based stones. Moreover, the manganese-dependent chemical mechanism employed by the enzyme has little precedent in known chemistry, and so its elucidation will add to knowledge concerning how the transition metal might participate in proton-coupled electron transfer to yield reactive radical intermediates that permit cleavage of the chemically inert C-C bond of oxalate. In our first specific aim, proposals for the catalytic mechanism of OxDC-catalyzed decarboxylation will be tested using advanced computational methods, X-ray crystallography, and the kinetic and spectroscopic characterization of a series of site-directed OxDC mutants. More specifically, we will pursue X-ray crystallographic studies aimed at obtaining detailed structural information on how oxalate is bound within the active site, the number of catalytically active sites in the enzyme, and the mode of dioxygen binding when the OxDC/oxalate complex is turning over under aerobic conditions. In addition, DFT and DFT/MM calculations will be carried out to assess whether hypothetical intermediates, and their associated transition states, are consistent with the kinetic properties of OxDC. Finally, the kinetic properties of site-specific mutants of the enzyme will be measured to delineate their functional roles in catalysis and/or active site dynamics. These findings will be correlated with predictions made on the basis of X-ray crystallography and computational studies, and a key goal will be to examine the extent to which Mn(III) and Mn(IV) mediate catalysis. The second specific aim will focus on understanding how the protein environment can modulate the intrinsic chemical reactivity of the Mn(II) center(s) in bacterial OxDC. Thus, the similarity of the Mn-binding motifs observed in plant oxalate oxidases (OxOx) and OxDC raises the question of how Mn(II) can be coordinated by identical ligands but catalyze different chemical transformations of the same substrate in each of the two enzymes. Systematic biophysical, isotope effect and computational studies of a series of OxOx/OxDC chimeras will be undertaken to validate existing hypotheses concerning the molecular basis for the observation that changes to a critical active site loop abolish decarboxylative activity with concomitant gain of oxidative function. Finally, in the third aim, we will investigate the ability of OxDC to dissolve human, calcium oxalate-based kidney stones in various types of salt-containing solutions so as to provide a basis for subsequent use of the enzyme in clinical applications.
Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human diseases associated with the accumulation of this compound in the blood and/or urine, including the formation of kidney stones. Our research group has played a leading role in characterizing the structure and catalytic mechanism of oxalate decarboxylase (OxDC), an oxalate-metabolizing enzyme that is present in fungi and some bacteria. In addition to providing new insights into the molecular mechanism by which OxDC can catalyze cleavage of the chemically unreactive C-C bond in oxalate, this project will also provide detailed information on how (i) protein environment can modulate transition metal chemistry, and (ii) amino acid mutations can be used to evolve new enzyme activities. Experiments will also be undertaken to assess the feasibility of employing OxDC in future, long-term translational research studies aimed at developing novel therapies for the clinical treatment and/or prevention of oxalate-related disease.
|Campomanes, Pablo; Kellett, Whitney F; Easthon, Lindsey M et al. (2014) Assigning the EPR fine structure parameters of the Mn(II) centers in Bacillus subtilis oxalate decarboxylase by site-directed mutagenesis and DFT/MM calculations. J Am Chem Soc 136:2313-23|
|Kellett, Whitney F; Brunk, Elizabeth; Desai, Bijoy J et al. (2013) Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase. Biochemistry 52:1842-4|
|Gonzalez, Ricardo D; Whiting, Bryant M; Canales, Benjamin K (2012) The history of kidney stone dissolution therapy: 50 years of optimism and frustration with renacidin. J Endourol 26:110-8|
|Saylor, Benjamin T; Reinhardt, Laurie A; Lu, Zhibing et al. (2012) A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase. Biochemistry 51:2911-20|
|Moussatche, Patricia; Angerhofer, Alexander; Imaram, Witcha et al. (2011) Characterization of Ceriporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris. Arch Biochem Biophys 509:100-7|
|Imaram, Witcha; Saylor, Benjamin T; Centonze, Christopher P et al. (2011) EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction. Free Radic Biol Med 50:1009-15|
|Moral, Mario E G; Tu, Chingkuang; Imaram, Witcha et al. (2011) Nitric oxide reversibly inhibits Bacillus subtilis oxalate decarboxylase. Chem Commun (Camb) 47:3111-3|
|Toyota, Cory G; Berthold, Catrine L; Gruez, Arnaud et al. (2008) Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase. J Bacteriol 190:2556-64|
|Berthold, Catrine L; Toyota, Cory G; Richards, Nigel G J et al. (2008) Reinvestigation of the catalytic mechanism of formyl-CoA transferase, a class III CoA-transferase. J Biol Chem 283:6519-29|
|Svedruzic, Drazenka; Liu, Yong; Reinhardt, Laurie A et al. (2007) Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis. Arch Biochem Biophys 464:36-47|
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