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Johannes Wostemeyer - One of the best experts on this subject based on the ideXlab platform.
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correlation between sequence structure and function for trisporoid processing proteins in the model zygomycete Mucor Mucedo
Journal of Theoretical Biology, 2013Co-Authors: Sabrina Ellenberger, Stefan Schuster, Johannes WostemeyerAbstract:Terpenoids, steroids, carotenoids, phytoenes and other chemically related substance groups fulfill multiple functions in all realms of the organismic world. This analysis focuses on trisporoids that operate as pheromones in the phylogenetically ancient fungal group of Mucoralean zygomycetes. Trisporoids serve as pheromones for recognizing complementary mating partners and for inducing the differentiation program towards sexual spore formation. Trisporoids are synthesized by oxidative degradation of β-carotene. Structurally, they are related to retinoids in mammals and abscisic acid in vascular plants. In order to evaluate evolutionary relationships between proteins involved in trisporoid binding and also for checking possibilities to recognize functionally related proteins by sequence and structure comparisons, we compared representative proteins of different origins. Towards this goal, we calculated three-dimensional structures for 4-dihydromethyltrisporate dehydrogenase (TSP1) and 4-dihydrotrisporin dehydrogenase (TSP2), the two proteins involved in trisporic acid synthesis that have unequivocally been correlated with their catalytic function for the model zygomycete Mucor Mucedo. TSP1 is an aldo-keto reductase with a TIM-barrel structure, TSP2 belongs to short-chain dehydrogenases, characterized by a Rossmann fold. Evidently, functional conservation, even implying very similar substrates and identical cosubstrates of enzymes in a single organism, turns out to be essentially independent of basic protein structure. The binding sites for NADP and trisporoid ligands in the proteins were determined by docking studies, revealing those regions affecting substrate specificity. Despite the pronounced differences in amino acid sequence and tertiary structure, the surfaces around the active sites are comparable between TSP1 and TSP2. Two binding regions were identified, one sterically open and a second closed one. In contrast to TSP1, all docking models for TSP2 place the trisporoid into the second, channel-like region.
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the mating related loci sexm and sexp of the zygomycetous fungus Mucor Mucedo and their transcriptional regulation by trisporoid pheromones
Microbiology, 2012Co-Authors: Jana Wetzel, Anke Burmester, Melanie Kolbe, Johannes WostemeyerAbstract:The putative mating type locus of Mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor Mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. Mucedo. The sex loci in M. Mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.
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4 dihydrotrisporin dehydrogenase an enzyme of the sex hormone pathway of Mucor Mucedo purification cloning of the corresponding gene and developmental expression
Eukaryotic Cell, 2009Co-Authors: Jana Wetzel, Christine Schimek, Anke Burmester, Olaf Scheibner, Johannes WostemeyerAbstract:The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-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.
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4 dihydromethyltrisporate dehydrogenase an enzyme of the sex hormone pathway in Mucor Mucedo is constitutively transcribed but its activity is differently regulated in and mating types
Fungal Genetics and Biology, 2005Co-Authors: Christine Schimek, Annett Petzold, Kornelia Schultze, Jana Wetzel, Frank Wolschendorf, Anke Burmester, Johannes WostemeyerAbstract:4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor Mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.
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biological activity of trisporoids and trisporoid analogues in Mucor Mucedo
Phytochemistry, 2005Co-Authors: Doreen Schachtschabel, Christine Schimek, Johannes Wostemeyer, Wilhelm BolandAbstract:Abstract In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of structurally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete structural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor Mucedo . The activity screening contributed to establish general structure–function relationships for trisporoid action. Most crucial for activity were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The overall biological activity apparently results from the combination of the various structural elements.
Sirma Yegin - One of the best experts on this subject based on the ideXlab platform.
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a thermolabile aspartic proteinase from Mucor Mucedo dsm 809 gene identification cloning and functional expression in pichia pastoris
Molecular Biotechnology, 2013Co-Authors: Sirma Yegin, Marcelo FernandezlahoreAbstract:In this study, the cDNA encoding the aspartic proteinase of Mucor Mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.
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purification structural characterization and technological properties of an aspartyl proteinase from submerged cultures of Mucor Mucedo dsm 809
Food Chemistry, 2012Co-Authors: Sirma Yegin, Yekta Goksungur, Marcelo FernandezlahoreAbstract:Abstract An extracellular aspartyl proteinase from Mucor Mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7 kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. Mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20 mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40 °C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55 °C for 10 min. These results indicate that the milk-clotting enzyme from M. Mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing.
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cloning and expression of the aspartic proteinase from Mucor Mucedo structural characterization and technological properties in comparison with the native enzyme
2011Co-Authors: Sirma YeginAbstract:This project explored the suitability of the aspartic proteinase from Mucor Mucedo DSM 809 as novel microbial rennet. The native proteinase was produced via submerged cultivation in shaking flasks. Under optimized conditions, the peak of production (130 U/ml) was observed after 48 h of cultivation at 24°C and 220 rpm. The chromatographically purified enzyme depicted a molecular weight (MW) of 32.7 kDa, an isoelectric point (pI) value of 4.29, and no evidence of N-linked glycosylation was found. When acting on milk to provoke curd formation, the M. Mucedo DSM 809 proteinase showed maximum potency at pH 5.0 and at 40°C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55°C for 10 min. The cDNA encoding the aspartic proteinase of M. Mucedo has been identified (GenBank accession # JN660818). The proteinase gene was cloned into pGAPZαA vector and expressed in Pichia pastoris X-33. Maximum enzyme production was observed when initial medium pH was 3.5 at 20°C and utilizing 4% glucose as a carbon and energy source. The enzyme was secreted in glycosylated form; two potential N-glycosylation sites were elucidated by site directed mutagenesis. All of the generated mutants have exhibited increase in milk clotting activity. The maximum activity obtained was 624 U/ml. Effect of copy number of the expression cassette on the activity has been discussed. Thermal stability studies of the purified proteins revealed that there was almost no effect of glycosylation on the thermal stability of the enzymes. The ratio of milk clotting activity to proteolytic activity for the unglycosylated enzymes was almost 3-fold higher than the glycosylated counterparts. The M. Mucedo proteinase / P. pastoris expression system has potential for the industrial production of a novel milk-clotting enzyme for cheese manufacturing.
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production of extracellular aspartic protease in submerged fermentation with Mucor Mucedo dsm 809
African Journal of Biotechnology, 2010Co-Authors: Sirma Yegin, Marcelo Fernandezlahore, Ulgar Guvenc, Yekta GoksungurAbstract:Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor Mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h of cultivation at 24°C in Erlenmeyer flasks. The optimized initial pH and shaking speed for enzyme production were 4.5 and 220 rpm, respectively. Glucose at a concentration of 1% (w/v) was the best carbon source for the production of enzyme among the carbohydrates examined (glucose, fructose, lactose, maltodextrin ). On the other hand casein at a concentration of 0.5% (w/v) was the selected nitrogen source in the media formulation. Under optimized conditions enzyme levels reached 130 SU per ml fermentation broth. The inoculum type and size has also affected biomass production and the biosynthesis of the enzyme. The preferred method was the inoculation of the culture media with spores at a total load of 6x10 5 spores per flask. Key words : Milk clotting enzyme, Aspartic protease, Mucor Mucedo, Sub-merged fermentation.
Marcelo Fernandezlahore - One of the best experts on this subject based on the ideXlab platform.
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a thermolabile aspartic proteinase from Mucor Mucedo dsm 809 gene identification cloning and functional expression in pichia pastoris
Molecular Biotechnology, 2013Co-Authors: Sirma Yegin, Marcelo FernandezlahoreAbstract:In this study, the cDNA encoding the aspartic proteinase of Mucor Mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.
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purification structural characterization and technological properties of an aspartyl proteinase from submerged cultures of Mucor Mucedo dsm 809
Food Chemistry, 2012Co-Authors: Sirma Yegin, Yekta Goksungur, Marcelo FernandezlahoreAbstract:Abstract An extracellular aspartyl proteinase from Mucor Mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7 kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. Mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20 mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40 °C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55 °C for 10 min. These results indicate that the milk-clotting enzyme from M. Mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing.
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production of extracellular aspartic protease in submerged fermentation with Mucor Mucedo dsm 809
African Journal of Biotechnology, 2010Co-Authors: Sirma Yegin, Marcelo Fernandezlahore, Ulgar Guvenc, Yekta GoksungurAbstract:Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor Mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h of cultivation at 24°C in Erlenmeyer flasks. The optimized initial pH and shaking speed for enzyme production were 4.5 and 220 rpm, respectively. Glucose at a concentration of 1% (w/v) was the best carbon source for the production of enzyme among the carbohydrates examined (glucose, fructose, lactose, maltodextrin ). On the other hand casein at a concentration of 0.5% (w/v) was the selected nitrogen source in the media formulation. Under optimized conditions enzyme levels reached 130 SU per ml fermentation broth. The inoculum type and size has also affected biomass production and the biosynthesis of the enzyme. The preferred method was the inoculation of the culture media with spores at a total load of 6x10 5 spores per flask. Key words : Milk clotting enzyme, Aspartic protease, Mucor Mucedo, Sub-merged fermentation.
Christine Schimek - One of the best experts on this subject based on the ideXlab platform.
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4 dihydrotrisporin dehydrogenase an enzyme of the sex hormone pathway of Mucor Mucedo purification cloning of the corresponding gene and developmental expression
Eukaryotic Cell, 2009Co-Authors: Jana Wetzel, Christine Schimek, Anke Burmester, Olaf Scheibner, Johannes WostemeyerAbstract:The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-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.
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4 dihydromethyltrisporate dehydrogenase an enzyme of the sex hormone pathway in Mucor Mucedo is constitutively transcribed but its activity is differently regulated in and mating types
Fungal Genetics and Biology, 2005Co-Authors: Christine Schimek, Annett Petzold, Kornelia Schultze, Jana Wetzel, Frank Wolschendorf, Anke Burmester, Johannes WostemeyerAbstract:4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor Mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.
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biological activity of trisporoids and trisporoid analogues in Mucor Mucedo
Phytochemistry, 2005Co-Authors: Doreen Schachtschabel, Christine Schimek, Johannes Wostemeyer, Wilhelm BolandAbstract:Abstract In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of structurally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete structural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor Mucedo . The activity screening contributed to establish general structure–function relationships for trisporoid action. Most crucial for activity were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The overall biological activity apparently results from the combination of the various structural elements.
Anke Burmester - One of the best experts on this subject based on the ideXlab platform.
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the mating related loci sexm and sexp of the zygomycetous fungus Mucor Mucedo and their transcriptional regulation by trisporoid pheromones
Microbiology, 2012Co-Authors: Jana Wetzel, Anke Burmester, Melanie Kolbe, Johannes WostemeyerAbstract:The putative mating type locus of Mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor Mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. Mucedo. The sex loci in M. Mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.
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4 dihydrotrisporin dehydrogenase an enzyme of the sex hormone pathway of Mucor Mucedo purification cloning of the corresponding gene and developmental expression
Eukaryotic Cell, 2009Co-Authors: Jana Wetzel, Christine Schimek, Anke Burmester, Olaf Scheibner, Johannes WostemeyerAbstract:The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-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.
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4 dihydromethyltrisporate dehydrogenase an enzyme of the sex hormone pathway in Mucor Mucedo is constitutively transcribed but its activity is differently regulated in and mating types
Fungal Genetics and Biology, 2005Co-Authors: Christine Schimek, Annett Petzold, Kornelia Schultze, Jana Wetzel, Frank Wolschendorf, Anke Burmester, Johannes WostemeyerAbstract:4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor Mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.
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4 dihydromethyltrisporate dehydrogenase from Mucor Mucedo an enzyme of the sexual hormone pathway purification and cloning of the corresponding gene
Microbiology, 1996Co-Authors: Katrin Czempinski, Johannes Wostemeyer, Volker Kruft, Anke BurmesterAbstract: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.