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United States Department of Agriculture

Agricultural Research Service

Title: Expression and Assembly of the Branched-Chain Alpha-Ketoacid Dehydrogenase Enzyme

Authors
item Mooney, B - UNIV OF MISSOURI
item Miernyk, Jan
item Randall, D - UNIV OF MISSOURI

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 3, 2002
Publication Date: N/A

Technical Abstract: The branched chain alpha-ketoacid dehydrogenase complex (BCKDC) is a multi-enzyme complex that catalyzes irreversible oxidative decarboxylation of the alpha-ketoacids derived from transamination of the branched-chain amino acids valine, leucine, and isoleucine. The BCKDC comprises three component enzymes, E1, E2, and E3. The E1 enzyme catalyses the first step in the BCKDC reaction. Two subunits, alpha and beta, form the E1 enzyme. The goal of the research was to determine the structure of Arabidopsis thaliana E1. Two constructs were designed to facilitate co-expression of the A. thaliana E1a and E1b subunits in bacteria. Recombinant E1b contains a six-His tag, while recombinant E1a has no affinity tags. E1a and E1b were co-expressed in Escherichia coli BL21-DE3 cells. When the E1b subunit was purified from clarified homogenates using Ni-NTA affinity chromatography, the E1a subunit co-purified, demonstrating the assembly of the E1 enzyme. Co-expression of the chaperonins GroEL and GroES improved the yield of recombinant E1. The molecular mass of E1, determined using size-exclusion chromatography, confirmed the formation of an a2b2 heterotetramer. Whereas the amino acid sequences of the plant E1 subunits are less than 40% identical to mammalian E1, the structure of the E1 enzyme is conserved across kingdoms.

Last Modified: 7/22/2014
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