4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo : Purification, Cloning of the Corresponding Gene, and Developmental Expression

Abstract

The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.