Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria

J Biochem Mol Toxicol. 2003;17(1):1-6. doi: 10.1002/jbt.10056.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • COS Cells
  • Carboxylic Acids / chemistry
  • Carboxylic Acids / metabolism
  • Cattle
  • Coenzyme A Ligases / genetics*
  • Coenzyme A Ligases / metabolism
  • DNA, Complementary / genetics*
  • DNA, Complementary / isolation & purification
  • Humans
  • Immunohistochemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Ligases / genetics
  • Mitochondria, Liver / enzymology*
  • Mitochondria, Liver / genetics
  • Molecular Sequence Data
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Xenobiotics / metabolism*

Substances

  • Carboxylic Acids
  • DNA, Complementary
  • Isoenzymes
  • Xenobiotics
  • Ligases
  • Coenzyme A Ligases