Molecular cloning, expression in Escherichia coli, and characterization of a novel L-3-hydroxyacyl coenzyme A dehydrogenase from pig liver.
A novel L-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized. This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties. Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme. In addition, there are substitutions of four single residues. The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths. The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional L-3-hydroxyacyl-CoA dehydrogenases in pig. High level expression of mitochondrial L-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important beta-oxidation enzyme. There is substantial homology between pig liver dehydrogenase and various multifunctional beta-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1-3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases.
He
X Y
XY
Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA.
Yang
S Y
SY
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine / Q000235:genetics*
D007527:Isoenzymes; D011994:Recombinant Proteins; D015094:3-Hydroxyacyl CoA Dehydrogenases