4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.
We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.
Czempinski
K
K
Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, FSU Jena, Neugasse, Germany.
Kruft
V
V
Wöstemeyer
J
J
Burmester
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D001330:Electronic Data Processing; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004790:Enzyme Induction; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008969:Molecular Sequence Data; D009089:Mucor / Q000235:genetics*; D009252:NADPH Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D010452:Peptide Biosynthesis; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D005231:Fatty Acids, Unsaturated; C004574:trisporic acid; D009252:NADPH Dehydrogenase; D010450:Endopeptidases