Chen, Hui , Kim, Hyun Uk , Shockey, Jay , Browse, John .
Biochemical approaches for the identification of the functions of CoA ligases in Arabidopsis.
Coenzyme A (CoA) ligases catalyze the activation of many carboxylic acids to the corresponding CoA derivatives through a two-step mechanism involving the conversion of the carboxylate and ATP to an enzyme-bound carboxyl-AMP intermediate with the release of PPi and then the formation of acyl-CoA with the release of AMP. CoA ligases and some related proteins are characterized by a highly conserved 12-amino-acid sequence that forms a core of the so-called AMP-binding motif (PROSITE PS00455). Based upon this conserved sequence signature, we identify a family of 44 genes in Arabidopsis that encode CoA ligases. Currently, about half of the 44 genes have known biochemical functions. We are using biochemical approaches to functionally characterize the remaining CoA ligase enzymes. Most of the proteins have been expressed in E. coli as polyhistidine fusions and purified to near-homogeneity. Both photospectrometer-based coupled-enzyme assays and HPLC-based assays have been developed for screening the activity of recombinant proteins using a collection of various carboxylic acids as potential substrates. Second we have developed a GC/MS method to profile the organic acid composition both in wild-type plant and in various homozygous mutants defect in one or two CoA ligase genes. To this end, we have identified AAE13 (At3g16170) as a novel malonyl-CoA ligase which utilizes malonic acid to synthesize malonyl-CoA and we have also detected the significant accumulation of free malonic acid in seedlings of one aae13 mutant but not in wild type seedlings. This is the first report of cloning a malonyl-CoA ligase gene from plant kingdom. This work is supported by NSF Arabidopsis 2010 project grant no. 0420199.
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1 - Washington State University, Institute of Biological Chemistry, Pullman, Washington, 99164, USA
2 - Washington State University, Institute of Biological Chemistry
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM