The Cloning, Expression, Purification And The Study Of The Catalytic Properties Of LCAD And ACAD9

Mitchondrial fatty acid β-oxidation provides a major energy-source in themetabolic process in heart and skeletal muscle. Especially during fasting or starvationperiod, ketone bodies produced by mitochondrial fatty acid oxidation in the liverbecome the major source of the body’s energy need. The acyl-CoA dehydrogenases(ACADs) are mitochondrial flavor-enzymes, catalyze the first reaction of themitochondrial β oxidation. There are11members in the family of ACADs, five ofthem catalyze the β oxidation of various chain length of straight-chain fatty acid:short-chain acyl-CoA dehydrogenase(SCAD), long-chain acyl-CoA dehydrogenase(LCAD), very long-chain acyl-CoA dehydrogenase(VLCAD) and acyl-CoAdehydrogenase9(ACAD9). They are different in molecular weight, structure,distribution and features. ACAD9and VLCAD are dimer, binding to themitochondrial inner membrane, and are difficult for solubilization, while other threeACADs are homotetramers in the mitochondrial matrix.LCAD and ACAD9are important members of the ACADs family, catalyzing thefirst dehydrogenation reaction of the mitochondrial β oxidation. Palmitoyl-CoA(C16-CoA) and stearoyl-CoA(C18-CoA) are the most abundant fatty acidcompound of plant and animal tissue, mainly catalyzed by LCAD、 ACAD9andVLCAD. In this research, the genes of LCAD and ACAD9were cloned,overexpressed and purified by one-step affinity chromatography. The molecularweights of the LCAD and ACAD9protein are45kDa and66.6kDa, respectively,containing a molecular of FAD. Through sequence aligment and LCADthree-dimensional structure simulation, we found the R317and Q385are highlyconserved, they bound with neighbouring FAD through hydrogen, G389and K419formed an important salt bridge between the two pairs of dimmers of the tetrameric.We constructed the mutant expression plasmid (E389A, E389D, E389Q) of the proteinusing site-directed mutagenesis, overexpressed in E.Coil and purified by one-stepaffinity chromatography. Results of the UV-Vis scanning and activity test confirmedthat residues Glu389of ACAD9were active-site residues. The carboxyl of Glu389takes the hydrogen on the α carbon atoms, thus transfering the hydrogen atoms toFAD. The electron on the substrate rearranged, forming the α-β Enoyl-CoA.