Probing the mechanism of Bacillus subtilis oxalate decarboxylase

dc.contributor.advisorRichards, Nigel G.
dc.contributor.authorZhu, Wen
dc.contributor.otherLi, Lei
dc.contributor.otherShah, Kavita
dc.contributor.otherMesecar, Andrew
dc.contributor.otherLong, Eric C.
dc.date.accessioned2016-06-22T20:42:38Z
dc.date.available2016-06-22T20:42:38Z
dc.date.issued2015-12-01
dc.degree.date2015en_US
dc.degree.disciplineChemistry & Chemical Biologyen
dc.degree.grantorPurdue Universityen_US
dc.degree.levelPh.D.en_US
dc.descriptionIndiana University-Purdue University Indianapolis (IUPUI)en_US
dc.description.abstractOxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.en_US
dc.identifier.doi10.7912/C2WS3K
dc.identifier.urihttps://hdl.handle.net/1805/10118
dc.identifier.urihttp://dx.doi.org/10.7912/C2/2259
dc.language.isoen_USen_US
dc.subject.lcshBacillus subtilis -- Research -- Physiologyen_US
dc.subject.lcshDecarboxylases -- Researchen_US
dc.subject.lcshOxalates -- Oxidation -- Researchen_US
dc.subject.lcshProteolytic enzymes -- Physiological effecten_US
dc.subject.lcshBacteria -- Physiologyen_US
dc.subject.lcshChemical kinetics -- Researchen_US
dc.subject.lcshMass spectrometryen_US
dc.subject.lcshChemistry, Analytic -- Researchen_US
dc.subject.lcshElectron paramagnetic resonance spectroscopyen_US
dc.subject.lcshCatalysisen_US
dc.subject.lcshX-ray crystallographyen_US
dc.subject.lcshAminesen_US
dc.titleProbing the mechanism of Bacillus subtilis oxalate decarboxylaseen_US
dc.typeThesisen
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