Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Basil J. Nikolau
Acetyl-CoA Carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl-CoA. Dicot plants have a homomeric ACC (hmACC) located in the cytosol responsible for the generation of malonyl-CoA pools for fatty acid elongation and secondary metabolites such as isoprenoids. Dicots utilize a heteromeric ACC (htACC) consisting of four subunits which as a holo-htACC generate malonyl-CoA for de novo fatty acid biosynthesis in the plastid. All ACC contain three functional domains. Biotin carboxylase (BC) catalyzes the ATP dependent carboxylation of biotin while carboxyltransferase (CT) catalyzes the transfer of the carboxyl group from biotin to acetyl-CoA. A biotin carboxyl carrier protein (BCCP) contains the cofactor biotin that is covalently linked to a conserved lysine residue and mediates between both half reactions. Arabidopsis thaliana (Arabidopsis) htACC contains natural structural diversity. Two isoforms of BCCP have been identified (CAC1A, At5g16390 encodes BCCP1, CAC1B, At5g15530 encodes BCCP2). In addition to BCCP isoforms, RNA-editing within the plastome encoded ACCD (AtCg00500 encoding CTβ) results in a serine to leucine point mutation at amino acid residue #265. Chapter II reports advancements made in the understanding of the structural diversity within Arabidopsis htACC in both BCCP and CTβ achieved through a combination of techniques including in silico molecular modeling, non-denaturing (native) polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography, circular dichroism, analytical ultracentrifugation, and mass spectrometry. Additionally, an evolutionarily related biotin-dependent carboxylase, 3-methylcrotonyl-CoA carboxylase (MCC), from Arabidopsis involved in leucine catabolism is included in this study. MCC is composed of two subunits (MCCA, encoded by MCCA (At1g03090) and MCCB, encoded by MCCB (At4g34030)). MCCA contains both BC and BCCP domains while MCCB contains CT functionality. MCC is of interest because it may contain a mixture of two forms of MCCA that are the result of alternative splicing of RNA from the gene MCCA that leads to either the inclusion or exclusion of the sixty nucleotide long 6th exon in Arabidopsis (MCCAL and MCCAS respectively). To date, little is known of the functional repercussions of the genetic diversity found in htACC and MCC from Arabidopsis. Chapter III describes the use of heterologous expression in E. coli to generate kinetically active subunits as determined by the use of traditional coupling assays. Additionally, BC substrate binding and characterization of BCCP1 and BCCP2 interaction with BC was studied using Differential Scanning Fluorimetry (DSF).
Bryon A. Upton
Upton, Bryon A., "Biochemical characterization of the biotin-dependent carboxylases, acetyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase" (2014). Graduate Theses and Dissertations. 13897.