Investigating the Mechanisms and Specificities of BphI-BphJ, an Aldolase-Dehydrogenase Complex From Burkholderia xenovorans LB400

dc.contributor.advisorSeah, Stephen YK
dc.contributor.authorBaker, Perrin
dc.date.accessioned2012-05-11T14:28:04Z
dc.date.available2012-05-11T14:28:04Z
dc.date.copyright2012-05
dc.date.created2012-05-02
dc.date.issued2012-05-11
dc.degree.departmentDepartment of Molecular and Cellular Biologyen_US
dc.degree.grantorUniversity of Guelphen_US
dc.degree.nameDoctor of Philosophyen_US
dc.degree.programmeMolecular and Cellular Biologyen_US
dc.descriptionChapter 3 - Reprinted (adapted) with permission from Baker, P., Carere, J., and Seah, S. Y. (2011) Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI, Biochemistry 50: 3559-3569. Copyright (2011) American Chemical Society. Chapter 4 - Reprinted (adapted) with permission from Baker, P., and Seah, S. Y. (2012) Rational design of stereoselectivity in the class II pyruvate aldolase BphI, J Am Chem Soc 134: 507-513. Copyright (2012) American Chemical Society. Chapter 6 - Reprinted (adapted) with permission from Baker, P., Hillis, C., Carere, J., and Seah, S. Y. (2012) Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes, Biochemistry 51: 1942-1952. Copyright (2012) American Chemical Society.en_US
dc.description.abstractMicrobial degradation of aromatic hydrocarbons is imperative for maintaining the global carbon cycle and removing potentially toxic aromatic xenobiotics. This thesis focuses on the characterization of a pyruvate-specific class II aldolase (BphI) and acetaldehyde dehydrogenase (BphJ), the final two enzymes of the bph meta-cleavage pathway in Burkholderia xenovorans LB400. This pathway is responsible for the degradation of the industrial pollutant polychlorinated biphenyls (PCB) and therefore mechanistic characterization of these enzymes can be applied to improve pollutant degradation. BphI catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to pyruvate and an aldehyde while BphJ transforms aldehydes to acyl-CoA, using NAD+ and CoASH as cofactors. Size-exclusion chromatography was used to determine that the oligomeric unit of the BphI-BphJ complex is a heterotetramer. The aldolase BphI was shown to exhibit a compulsory order mechanism and utilize 4-hydroxy-2-oxoacids with an S configuration at C4. The generation of BphI active site variants allowed for the proposal of a catalytic mechanism and a greater understanding as to how stereospecificity occurs. Using steady-state kinetic assays, Arg-16 was demonstrated to be essential for catalysis. Molecular modeling of the substrate and pH dependency (wild-type pKa of ~7, lost in H20A and H20S variants) were used to identify His-20 as the catalytic base. Tyr-290 was originally proposed to be the catalytic acid. However, this was refuted as a Tyr-290 (Y290F) variant did not affect the catalytic efficiency of the enzyme. Instead, the variant was observed to exhibit a loss of stereochemical control. From the crystal structure of an orthologous aldolase-dehydrogenase complex, solvent isotope effect studies, and a proton inventory, a water molecule was implicated as the catalytic acid. Based on their position within the crystal structure, Leu-87 and Leu-89 were implicated in substrate specificity. Replacement of Leu-89 with alanine effectively increased the length of the active site, allowing for the accommodation of longer aldehyde substrates. In contrast, Leu-87 was responsible for hydrophobic stabilization of the C4-methyl of the substrate. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids. Polarimetric analysis confirmed that the double variants were able to synthesize 4-hydroxy-2-oxoacids of up to 8 carbons in lengths, which were of the opposite stereoisomer to those produced by the wild-type enzyme. Cys-131 was identified as the catalytic thiol that forms an acyl-enzyme intermediate in the dehydrogenase, BphJ. This enzyme was shown to exhibit similar specificity constants for acetaldehyde and propionaldehyde and utilize aliphatic aldehydes from two to five carbons in length as substrates. The enzyme was able to use either NAD+ or NADP+ as the cofactor. Finally, we demonstrated that aldehydes produced in the aldolase reaction are not released into the bulk solvent but are channeled directly to the dehydrogenase, providing the first biochemical determination of substrate channeling in any aldolase-dehydrogenase complex.en_US
dc.description.sponsorshipNational Science and Engineering Research Council of Canada
dc.description.sponsorshipOntario Graduate Scholarship in Science and Technology
dc.identifier.urihttp://hdl.handle.net/10214/3612
dc.language.isoenen_US
dc.publisherUniversity of Guelphen_US
dc.rights.licenseAll items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectsubstrate channelingen_US
dc.subjectaldolaseen_US
dc.subjectpyruvateen_US
dc.subjectacetaldehydeen_US
dc.subjectdehydrogenaseen_US
dc.subjectcatalytic mechanismen_US
dc.subjectmechanismen_US
dc.subjectenzymeen_US
dc.subjectstereospecificityen_US
dc.subjectstereochemical controlen_US
dc.subjectmutagenesisen_US
dc.subjectallosteryen_US
dc.subjectstructureen_US
dc.subjectfunctionen_US
dc.subjectsubstrate specificityen_US
dc.titleInvestigating the Mechanisms and Specificities of BphI-BphJ, an Aldolase-Dehydrogenase Complex From Burkholderia xenovorans LB400en_US
dc.typeThesisen_US

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