Studies of Escherichia coli Acetyl-CoA carboxylase
Acetyl-CoA carboxylase (ACC) initiates the first committed step of fatty acid synthesis. ACC is composed of three separate components: biotin carboxylase (BC), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). BC carries out the first half of the ACC reaction where it catalyzes the carboxylation of a biotin moiety covalently linked to the BCCP. BC exists as a dimer but mutated versions of BC deficient in dimerization were reported to retain most of their enzymatic activity in vitro. I report that two different mutants severely deficient in their ability of form dimers are unable to complement growth as the sole source of BC unless expressed at very high levels. The BC dimer mutants retained most of their activity in vitro even though their Kd had decreased hundreds fold. I functionally replaced the endogenous accC with plasmids expressing either wild type or one of the dimer mutants. While the dimer mutants were able to complement at high expression levels, they failed at the more modest levels which permitted growth with the wild type protein. Therefore BC must remain a dimer to fulfill its physiological function. CT carries out the second part of the ACC reaction where it transfers the biotin bound carboxyl group to acetyl-CoA in order to form malonyl-CoA which is destined for fatty acid synthesis. CT consist of a heterotetramer of AccA and AccD which in E. coli are encoded by structural genes at the opposite ends of the chromosome. Stoichiometric amounts of AccA and AccD are required and so the method to achieve this remains unclear. It was reported that the AccD subunit of CT is able to bind the transcripts of each subunit to allow for a degree of translational repression. Long stretches (600 bp) of naked RNA were required to induce binding in vitro and so the plausibility of this method of regulation was questioned. I have replicated these conditions in vivo using a multi plasmid dual promoter system which allows for measurement of subunit synthesis in the presence of varying quantities of CT tetramer. I report that increased levels of CT tetramer have no effect on translation of the CT subunit mRNAs. The PII nitrogen regulation proteins from Arabidopsis were found to inhibit ACC activity in a manner sensitive to its natural substrate, 2-oxoglutarate (2-OG). Given the close relation of plant plastid fatty acid synthetic enzymes and those in bacteria it was logical to test for this effect in E. coli. I report that the E. coli PII protein GlnB causes inhibition of ACC activity which is then reversed by the addition of 2-OG. The two PII proteins of E. coli, GlnB and GlnK, were purified and then tested for inhibition with overexpressed ACC cell extract.
E. coli, Escherichia coli, Fatty acid synthesis, Acetyl-CoA Carboxylase (ACC), Biotin Carboxylase (BC), Carboxyltransferase (CT), Malonyl-CoA, PII, Dimerization, Translational Repression
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign