Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria.
The purification of xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM-A and HXM-B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N-terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM-A and HXM-B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full-length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64230-Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium-chain acyl-CoA synthetase) enzyme, 58.7% homology to the bovine XL-III XM-ligase, and 81.5% homology to the bovine XL-I XM-ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM-A.
Vessey
Donald A
DA
Department of Veterans' Affairs Medical Center, San Francisco, California 94121, USA.
Lau
Eva
E
Kelley
Michael
M
Warren
Robert S
RS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008025:Ligases / Q000235:genetics; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism*
D002264:Carboxylic Acids; D018076:DNA, Complementary; D007527:Isoenzymes; D015262:Xenobiotics; D008025:Ligases; D003066:Coenzyme A Ligases