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 ␤-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.The existence of a conserved system for sexual communication in zygomycetes had been assumed very early based on observations of the ability of mucoralean fungi belonging to different species to differentiate into early sexual morphological stages in cocultures on petri dishes, although without leading to zygospores (4, 7). These observations allowed consistent assignment of zygomycetes across species, genus, and family borders to (ϩ) or (Ϫ) mating types.In contrast to the modified peptide pheromones in asco-and basidiomycetes, the sexual reaction of zygomycetes is mediated by trisporic acid and its numerous derivatives and isomers. Trisporoids are involved in partner recognition, sexual morphogenesis, and feedback regulation of trisporoid synthesis. Especially in Mucor mucedo, they also induce the first discernible sexual differentiation structures, the zygophores. Physiological aspects of the trisporic acid system for sexual communication have recently been reviewed (27,38).Although the synthesis of trisporic acid from ␤-carotene (2) is still hypothetical in some parts of the pathway, the best and most consistent model assumes a shared and cooperative synthesis by both complementary mating types, accomplished by exchanging mating-type-specific precursors between complementary sexual partners (5,6,23,35,36). Altho...

The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones

“…These are synthesized along a cooperative biochemical pathway that requires both sexually complementary mating types to produce trisporoids at concentrations that are high enough to induce the sexual differentiation pathway (Wöstemeyer et al, 1995). For the (2) mating types of M. mucedo and Parasitella parasitica, we have shown previously that key functions of trisporic acid synthesis are not under the control of a common transcription factor, but instead are regulated post-transcriptionally and post-translationally Schultze et al, 2005;Wetzel et al, 2009). Provided the regulation of trisporoid synthesis is sufficiently tight to prevent sexual reactions in a single mating type, and to warrant it in co-cultures, there is no prior necessity for a master mating type locus or a general transcription factor.…”

“…Here, more attention has been given to the molecular biology of partner recognition that is mediated by trisporoid pheromones, synthesized by degradation of carotene. Some of the genes as well as the corresponding gene products necessary for synthesizing trisporic acid are known in detail (Czempinski et al, 1996;Wetzel et al, 2009;Burmester et al, 2007).…”

“…To date, one enzyme as well as the corresponding gene, i.e. carotene oxygenase (TSP3) as well as two (2) type-specific steps of this pathway have been identified and characterized in M. mucedo: the 4-dihydromethyltrisporate dehydrogenase (TSP1; Czempinski et al, 1996;Schimek et al, 2005) and the 4-dihydrotrisporin dehydrogenase (TSP2; Wetzel et al, 2009). …”

The Evolution of Sex: a Perspective from the Fungal Kingdom

“…In brief, (ϩ) strains produce 4-dihydromethyl trisporate, which is delivered into (Ϫ) strains. Interestingly, the key enzyme for this reaction, 4-dihydrotrisporin-dehydrogenase, exists in both mating types, and only enzyme activity is differentially regulated in the Mucorales fungus Mucor mucedo (305). The (Ϫ) strains then produce methyltrisporate from 4-dihydromethyl trisporate and finally form trisporic acid.…”

Early and Late Trisporoids Differentially Regulate β-Carotene Production and Gene Transcript Levels in the Mucoralean Fungi Blakeslea trispora and Mucor mucedo

“…1) from D'orenone (␤-apo-13-carotenone) to methyl trisporate in the trisporic acid biosynthetic pathway are known as trisporoids (17)(18)(19). Few enzymes are known so far, such as 4-dihydromethyltrisporate dehydrogenase (TSP1), 4-dihydrotrisporin dehydrogenase (TSP2) (20)(21)(22), and a putative esterase enzyme from the minus partner that converts methyl trisporate to trisporic acids in both homothallic Mucorales (Zygorhynchus moelleri) and heterothallic Mucorales (23). The high-mobility group (HMG) transcription factor genes sexM and sexP are associated with the minus and plus mating-type loci in P. blakesleeanus and M. mucedo (24,25).…”