cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233.
We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.
Nuttley
W M
WM
Department of Biochemistry, McMaster University, Hamilton, Ont., Canada.
Aitchison
J D
JD
Rachubinski
R A
RA
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D004271:DNA, Fungal / Q000235:genetics*; D004746:Enoyl-CoA Hydratase / Q000235:genetics*; D005796:Genes; D005800:Genes, Fungal; D006836:Hydro-Lyases / Q000235:genetics*; D007535:Isomerases / Q000235:genetics*; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D063994:Peroxisomal Bifunctional Enzyme; D011487:Protein Conformation; D019998:Racemases and Epimerases / Q000235:genetics*; D015183:Restriction Mapping
D003062:Codon; D004271:DNA, Fungal; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases; D019998:Racemases and Epimerases