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An Investigation of the Molecular Determinants of Substrate Channeling and Allosteric Activation in Aldolase-Dehydrogenase Complexes

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dc.contributor.advisor Seah, Stephen
dc.contributor.author Carere, Jason
dc.date.accessioned 2013-05-06T20:06:30Z
dc.date.available 2013-05-06T20:06:30Z
dc.date.copyright 2013-04
dc.date.created 2013-04-30
dc.date.issued 2013-05-06
dc.identifier.uri http://hdl.handle.net/10214/6623
dc.description.abstract The aldolase-dehydrogenase complex catalyzes the last two steps in the microbial meta-cleavage pathway of various aromatic compounds including polychlorinated biphenyls (bph pathway) and cholesterol (hsa pathway). The aldolase, BphI, cleaves 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. Linear aldehydes of up to six carbons long and branched isobutyraldehyde were directly channeled to the aldehyde dehydrogenase BphJ, via a molecular tunnel, with greater than 80% efficiency. The molecular tunnel is narrow in positions lined by Gly-322 and Gly-323 in the aldolase. BphI variants G322F, G322L and G323F were found to block aldehyde channeling. The replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies, thus disproving a previous hypothesis that hydrogen bonding between the Asn-170 and the nicotinamide cofactor induces the opening of the exit of the tunnel. The H20A and Y290F BphI variants displayed significantly reduced aldehyde channeling efficiencies indicating that these residues control the entry and exit of substrates and products from the aldolase reaction. The BphI reaction was activated by NADH binding to BphJ in the wild-type enzyme and channel blocked variants. Activation of BphI by BphJ N170A, N170D and I171A was decreased by ≥ 3-fold in the presence of NADH and ≥ 4.5-fold when BphJ was undergoing turnover. These results demonstrate that the dehydrogenase coordinates catalytic activity of BphI through allostery rather than through faster aldehyde release from substrate channeling. HsaF, an ortholog of BphI from Mycobacterium tuberculosis could be expressed as a soluble dimer, however HsaF was inactive in the absence of HsaG, a BphJ ortholog. Acetaldehyde and propionaldehyde were channeled directly to HsaG with similar efficiencies as in the BphI-BphJ system. The HsaF-HsaG complex was crystallized and its structure solved to a resolution of 1.93 Å. Substitution of Ser-41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km for dephospho-CoA, confirming its importance in interacting with the 3’- ribose phosphate of CoA. A second gene annotated as 4-hydroxy-2-oxopentanoic acid aldolase (Rv3469c) from M. tuberculosis was expressed, purified and found to possess oxaloacetate decarboxylase and not aldolase activity. en_US
dc.language.iso en en_US
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/2.5/ca/ *
dc.subject Aldolase en_US
dc.subject Dehydrogenase en_US
dc.subject Channeling en_US
dc.subject Crystallography en_US
dc.subject Allostery en_US
dc.subject Cholesterol en_US
dc.subject PCBs en_US
dc.title An Investigation of the Molecular Determinants of Substrate Channeling and Allosteric Activation in Aldolase-Dehydrogenase Complexes en_US
dc.type Thesis en_US
dc.degree.programme Molecular and Cellular Biology en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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