Search

The filamentous fungus Phycomyces blakesleeanus provides a renewable biosource of industrial high-value compounds such as carotenes, other isoprenoids (ubiquinone and sterols), organic acids and fatty acids. Several Phycomyces mutants involved in the formation of β-carotene are available. For example, the carA mutants have a leaky mutation in the phytoene synthase and produce significantly lower amounts of carotenes, while the carB and carR mutants produce phytoene and lycopene, respectively, due to a null mutation in the genes encoding the phytoene dehydrogenase and lycopene cyclase, respectively. The carS mutants are mutated in the gene encoding the oxygenase responsible for the conversion of β-carotene into apocarotenoids and, as a result, β-carotene accumulates. In order to ascertain further the biochemical changes arising in these potential industrial strains, a metabolite profiling workflow was implemented for Phycomyces. GC-MS and ultra-performance liquid chromatography-photodiode array platforms enabled the identification of over 100 metabolites in 11 carA, carB, carR and carS mutant strains and their wild-type comparator. All mutant strains possessed decreased TCA cycle intermediates, galactose, alanine and ribitol, while dodecanol and valine showed a general increase. As predicted, other terpenoid levels were affected in the carB, carR and carS mutants but not in the carA mutants. The global changes across intermediary metabolism of the mutants suggest that complex metabolic networks exist between intermediary and secondary metabolism or that other mutations beyond the carotene pathway may exist in these mutants. These data show the utility of the methodology in metabolically phenotyping Phycomyces strains with potential industrial exploitation.

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mixed cultures of strains of opposite sex of the Mucorales produce trisporic acids and other compounds arising from cleavage of β-carotene, some of which act as signals in the mating process. The genome of Phycomyces blakesleeanus contains five sequences akin to those of verified carotenoid cleavage oxygenases. All five are transcribed, three of them have the sequence traits that are considered essential for activity, and we have discovered the reactions catalysed by the products of two of them, genes carS and acaA. The transcripts of carS became more abundant in the course of mating, and its expression in β-carotene-producing Escherichia coli cells led to the formation of β-apo-12'-carotenal, a C₂₅ cleavage product of β-carotene. Joint expression of both genes in the same in vivo system resulted in the production of β-apo-13-carotenone, a C₁₈ fragment. In vitro, AcaA cleaved β-apo-12'-carotenal into β-apo-13-carotenone and was active on other apocarotenoid substrates. According to these and other results, the first reactions in the apocarotenoid pathway of Phycomyces are the cleavage of β-carotene at its C11'-C12' double bond by CarS and the cleavage of the resulting C₂₅-fragment at its C13-14 double bond by AcaA., (© 2011 Blackwell Publishing Ltd.)

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

D-erythroascorbate (D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate monoglucoside (D-EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and 1084 μg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds showed UV spectral properties and ionization constants similar to those of L-AA. D-EAAG was much more stable to aerobic oxidation than D-EAA and L-AA at acidic pH. D-EAAG is synthesized from D-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate donor with K(m) = 2.5 mM and 41.3 μM for D-EAA. This glucosyltransferase activity was maximal in the stationary growth phase in parallel with maximal production of D-EAAG. The presence of D-arabinose or D-arabinono-1,4-lactone in the culture medium produces the maximal accumulation of D-EAA and D-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds showed greater antioxidant activity than L-AA and other standard antioxidants, with a capacity similar to that of L-AA to inhibit the growth of Escherichia coli.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type., (2010 Elsevier Inc. All rights reserved.)

Background: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy., Methodology/principal Findings: Here we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently., Conclusions/significance: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events.

The stability of acetyl-CoA synthetases (ACS1 and ACS2) from P. blakesleeanus against temperature, urea and trypsin was studied and compared. Thermal inactivation of ACS1 was biphasic, while that of ACS2 was monophasic. The thermodynamic parameters calculated from the inactivation profiles show ACS2 to be a more thermostable enzyme than ACS1. The presence of ATP and Mg(2+) exerted a protective effect on both enzymes, and led to a marked increase in the E(a), DeltaH(not =), DeltaS(not =) and DeltaG(not =) values. ACS2 is also much more stable against denaturation with urea; the estimates of DeltaG(w) (free energy change for protein unfolding at zero denaturant concentration) were 9.4 kJ mol(-1) and 18.1 kJ mol(-1) for ACS1 and ACS2, respectively. Finally, a half-life of 44.5 min for ACS2 versus the 21 min for ACS1 indicates that ACS2 is more stable than ACS1 against digestion by trypsin. These results seem to show that ACS2 is more rigid overall than ACS1, which may be essential for preserving its catalytic activity in the stress situation in which it is expressed.

Applications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene beta-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to beta-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.

Two forms of acetyl-CoA synthetase (ACS1 and ACS2) have been detected in Phycomyces blakesleeanus. ACS1, encoded by the gene facA, was induced by acetate and repressed by glucose at the transcriptional level. ACS2, not encoded by the gene facA, was detected as a response to carbon starvation both in the wild type and in an facA(-) mutant. Both enzymes were purified and characterized. They can use acetate and propionate as substrates. ACS2 is a much more stable enzyme than ACS1. After 60 min incubation at 55 degrees C, ACS2 retained 50% of its activity whereas ACS1 only retained 3%. The optimum temperature was 50 degrees C for ACS2 and 30 degrees C for ACS1.

D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of l-ascorbate, suggesting that D-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed.

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a pce1 gene, was cloned by plaque hybridization with the codon usage-optimized rce1 gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The pec1 gene had an open reading frame of 1,038 nucleotides encoding an endoglucanase (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pce1 gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. nitens and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50 degrees C, the lowest among the family 45 endoglucanases.

The zygomycete Phycomyces blakesleeanus has a 30 Mb genome with a 35% content of guanine and cytosine (GC). We determined the GC content in Phycomyces genes and fragments of genes available in public databases, the frequency of nucleotides in each codon position, and the codon usage. We observed a difference of 18% between the GC content of protein-coding and non-coding DNA. This large difference allowed the visualization of protein-coding DNA by plotting the GC content along a segment of Phycomyces DNA. We have identified a high GC DNA segment linked to the pyrG genes of the zygomycete genera Phycomyces, Mucor, and Blakeslea that corresponds to the 3' end of the gene responsible for the protein kinase C.

Dormant spores of Phycomyces blakesleeanus contain a 37 kDa protein that is endogenously mono-ADP-ribosylated. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing and homology analysis. GAPDH enzymic activity changed dramatically upon spore germination, being maximal at stages where ADP-ribosylation was nearly undetectable. The presence of glyceraldehyde 3-phosphate in this reaction affected the [(32)P]ADP-ribosylation of the GAPDH. ADP-ribosylation of the GAPDH occurred by transfer of the ADP-ribose moiety from NAD to an arginine residue. A model for the regulation of GAPDH activity and its role in spore germination in P. blakesleeanus is proposed.

Blue light controls the development of sporangiophores in the zygomycete Phycomyces blakesleeanus Burgeff. Light represses the production of microsporangiophores and enhances the development of macrosporangiophores. Inhibition of the biosynthesis of tetrahydrobiopterin, a cofactor of NO synthase, inhibits this photomorphogenesis. Light induces production of citrulline from arginine in the mycelium and in sporangiophores. The citrulline-forming activity is dependent on NADPH, independent of calcium, and inhibited by NO synthase inhibitors. It is reduced in tetrahydrobiopterin-depleted mycelium. Light induces emission of NO from the developing fungus in the same order of magnitude as citrulline formation from arginine. The NO donor sodium nitroprusside can replace the light effect on sporangiophore development, and inhibitors of NO synthase repress it. We suggest that a fungal NO synthase is involved in sporangiophore development and propose its participation in light signaling.

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.

Carotenogenesis and morphogenesis represent two of the several responses sensitive to blue light which characterize the lower eukaryote Phycomyces blakesleeanus. Speculating that reversible phosphorylation may be an intracellular event beyond the photoperception step, we resorted to the use of first-choice inhibitors of protein phosphatases and protein kinases. The mycelial beta-carotene content of dark-grown cultures was induced by all agents administered, while the morphogenic output showed the typical trend effected by light only with one of the protein kinase inhibitors. Our data provide convincing evidence that protein phosphorylation plays a regulatory role in photocarotenogenesis and photomorphogenesis of Phycomyces. According to the model we propose, the putative signaling elements involved are anticipated to have a repressive function in the dark so that the responses are maintained in the "off" mode until the moment photon information has to flow through the regulatory circuit., (Copyright 1999 Academic Press.)

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Mg2+ and Mn2+ function with the same partial mixed-type activation/inhibition mechanism, in which the metal isocitrate complex is the true substrate of Phycomyces isocitrate lyase. Binding of Mg2+ or Mn2+ to the activation site normally contributes significantly to the mechanism of catalysis. Whereas both ions activate catalysis at pH 7.3, at pH 8.5, Mg2+ ions behaved as inhibitors (beta < 1) and Mn2+ ions continued to function as activators. The binding of Mg2+ or Mn2+ to the activator site is virtually independent of the pH value. The affinity of the non-activated form of the enzyme for the Mg(2+)-isocitrate complex decreased (Ksa increased 20-fold) as pH was raised, but for Mn2+ ions the affinity of the activated enzyme for the Mn(2+)-isocitrate complex decreased 86-fold. The ion moiety of the metal-ion-isocitrate complex appears to be involved in the formation of the active enzyme-substrate complex from the non-activated enzyme.

By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.

A soluble arginine-specific mono(ADP-ribosyl)transferase was detected in dormant spores of Phycomyces blakesleeanus. Soluble proteins incubated with [32P]NAD revealed, after a two dimensional electrophoretic separation, three major ADP-ribosylated substrates with molecular weights of 38, 37, and 36 kDa and pI values of 6.9, 8.1 and 4.6, respectively. The addition of MgCl2 stimulated the (ADP-ribosyl)transferase activity. This enzymatic activity was stimulated by 250 microM NO-releasing agent sodium nitroprusside and inhibited with 8 mM benzamide, 0.4 mM meta-IodoBenzylGuanidine (MIBG), and 0.5 mM novobiocin. The three ADP-ribosylation inhibitors affected the germination of Phycomyces spores. The concentrations necessary to inhibit 50% of the spore germination of Phycomyces were 0.05 mM, 0.2 mM, and 8 mM for novobiocin, MIBG, and benzamide, respectively. All the above inhibitors affected the germination process to the same extent, that is, they inhibited the tube protuberation, leaving the spores as swollen cells. These data suggest that ADP-ribosylation may be involved in the germination process of Phycomyces, particularly in germ-tube formation.

An acid protease has been found in the culture broth of Phycomyces blakesleeanus growing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease of Phycomyces migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A-Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70 degrees C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50 degrees C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-N02)-Arg-Leu as the substrate, the Km, kcat, and Vmax values were 8.78 microM, 1.25 s(-1), and 2.12 mumol min(-1) mg(-1), respectively. The protease was insensitive to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with a K(i) value of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase.

Two forms of NAD+-isocitrate dehydrogenase, named ICDH-1 and ICDH-2, have been identified and purified in Phycomyces blakesleeanus NRRL-1555(-). These enzymes forms may be separated by chromatography on DEAE-Sephacel. ICDH-2 induction was a response to the adaptation of Phycomyes growth on acetate as the carbon source. Both enzyme forms were octamers of 388 + or - 30 kDa with apparently identical subunits of 40.5 +/- 5 kDa, but they were distinguishable by their electrophoretic mobilities on polyacrylamide gel electrophoresis. Isoelectric pH values were 5.28 and 4.96 for ICDH-1 and ICDH-2, respectively. ICDH-2 was more stable to urea denaturation than ICDH-1. At pH 7.6, ICDH-1 showed a markedly sigmoidal kinetic behavior with respect to isocitrate. However, ICDH-1 and ICDH-2 showed hyperbolic kinetics with respect to NAD+. THe tribasic form of isocitrate (I3-) and its magnesium complex (MI-) are the true substrates for both enzyme forms. Kinetic data obtained with Mg2+ as a divalent cation for both enzyme forms are compatible with the kinetic mechanism proposed by Cohen and Colman (1974) [Eur. J. Biochem. 47, 35-45] but assuming some degree of interaction between binding sites for the active form of isocitrate. This report describes for the first time the existence of two forms of NAD+-isocitrate dehydrogenase in filamentous fungi. From the changes in activity levels for each form, during adaptation of Phycomyces to growth on acetate and taking into account the kinetic and regulatory properties of both enzyme forms, we discuss the role of ICDH-1 and ICDH-2 in the control of isocitrate flux in Phycomyces.

We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaxpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units. Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second-order rate constant of 18.58 M-1 s-1 (at pH 6.0 and 20 degrees C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N-carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O-carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N-carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data-protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.

This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth. This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins. However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P. blakesleeanus.

Progesterone hydroxylase cytochrome P-450 was purified to homogeneity from Phycomyces blakesleeanus microsomes by a four step procedure. An M(r) value of 60,000 was determined for this protein by SDS-PAGE. The DEAE-cellulose and Blue-1 MIMETIC affinity fractions gave major peaks at 452 nm in a dithionite-reduced, carbon monoxide, difference spectrum. NaIO4-dependent progesterone hydroxylation was obtained by the pure enzyme without NADPH and NADPH-cytochrome P-450 reductase. NADPH-dependent hydroxylation required the addition of other Phycomyces microsomal proteins present in the Blue-1 fraction.

Occurrence, biosynthesis and some functions of tetrahydrobiopterin (H4biopterin) in animals are well known. The biochemistry of H4biopterin in other organisms than animals was hitherto not widely investigated. Recently H4biopterin was found in the phytoflagellate Euglena gracilis, in the zygomycete Phycomyces blakesleeanus and in the ascomycete Neurospora crassa. In Euglena, Neurospora and Phycomyces the enzymatic activities of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are detectable and the biosynthesis follows the same steps as were shown for animals. The biosynthetic enzymes, however, show a much lower sensitivity to those inhibitors that act on vertebrate enzymes. 2,4-Diamino-6-hydroxypyrimidine as inhibitor of GTP cyclohydrolase I and N-acetylserotonin or N-methoxyacetylserotonin as inhibitors of sepiapterin reductase can decrease pteridine biosynthesis significantly, in vitro and in vivo. The apparent Km values are in general higher when compared with the respective animal enzymes. In Neurospora, the conversion of GTP to dihydroneopterin triphosphate was closely associated with subsequent production of 6-hydroxymethyl-7,8-dihydropterin due to the high activity of dihydroneopterin aldolase, different from all other tested organisms. Investigations involving inhibition of pteridine synthesis might be a useful tool for evaluating the hypothesis that pteridines in fungi and plants are co-chromophores of various blue light-dependent, flavin-containing photoreceptors.

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanus protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA- mutation of this organism. In P. blakesleeanus, the expression of facA is induced by acetate.

The carotenogenic enzyme phytoene dehydrogenase has been purified from the C9carR21(-) (lycopene-accumulating) mutant of the filamentous fungus Phycomyces blakesleeanus. Solubilization of the membrane-bound enzyme with 1% Tween-60 was followed by a 250-fold purification to homogeneity using polyethylene glycol precipitation, CM-Sepharose, gel filtration and isoelectric focusing. Multiple peaks of enzymic activity were found in eluates from ion-exchange and gel filtration chromatography, with the lowest molecular weight fraction having an apparent molecular mass of approx. 14 kDa. All active fractions catalyzed the dehydrogenation of 15-cis phytoene into all-trans lycopene, with a cis-trans isomerization occurring at phytofluene. Both NADP+ and FAD were required for the dehydrogenation reaction. The presence of > 0.5% Tween-60 was necessary to maintain enzymic activity, although in its absence lipids restored some activity. The enzyme could be stored for at least 6 weeks at -70 degrees C in the presence of 20% (v/v) glycerol.

Using polymerase chain reaction (PCR) primers to two highly conserved sequences within fungal chitin synthase (CHS)-encoding genes, an approximately 750-bp DNA fragment was amplified from genomic DNA of Phycomyces blakesleeanus. The amino acid sequence deduced from the nucleotide sequence of this fragment best matches the motifs found in class-II CHS. The fragment includes an approximately 160-bp region that likely is an intron. Southern hybridization of restriction enzyme-digested genomic DNA, using the PCR-amplified DNA as the probe, suggests that P. blakesleeanus contains additional CHS-encoding genes (CHS). To our knowledge, this is the first report on the detection of a CHS gene in a Zygomycete fungus. These studies represent a major step toward exploring the molecular mechanisms of CHS regulation in Phycomyces. The prospects are exciting, since CHS is implicated to play a central role in the sensory responses of P. blakesleeanus involving growth modulations.

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 microM nikkomycin, chsA mutants grew reasonably well in the presence of 50 microM nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased Ka for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.

Isocitrate lyase from the mycelium of Phycomyces blakesleeanus was inactivated with thiol-reactive reagents, 5,5'-dithiobis-(2-nitrobenzoic)acid, p-hydroxymercuribenzoic acid, N-ethylmaleimide or iodoacetate, at pH 6.8 and 25 degrees C. In all cases the inactivation is characterized by a biphasic kinetic profile. The rapid initial phase of inactivation does not increase linearly with increasing reagent concentration, but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. Re-activation of the enzyme was observed under thiol excess treatment. The pH dependence of the initial phase of inactivation suggests that a group on the enzyme with pKa = 6.8 is being modified. The effect of ligands was tested on the inactivation reaction. Mg(2+)-Ds-isocitrate and Ds-isocitrate provided total protection, whereas Mg2+ ions, succinate and oxalate provided only partial protection of the enzyme against inactivation. On the basis of these results, we would suggest that the thiol-reactive reagents modify at least one thiol group crucial for the enzymatic activity and probably located in the interface between succinate and glyoxylate subsite.

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.

The existence and the mode of operation of certain enzyme aggregates may be established from the concentrations of intermediates measured in the presence of specific inhibitors. beta-Carotene, the most abundant carotenoid pigment in the fungus Phycomyces blakesleeanus, arises from ring formation at both ends of lycopene. The inhibitors nicotine, imidazole, alpha-picoline, and 2-(4-chlorophenylthio)triethylamine lead to the simultaneous accumulation of lycopene, beta-carotene, and the one-ring intermediate gamma-carotene. The quantitative analytical values obey precise mathematical relationships: those expected from the operation of an enzyme aggregate with two cyclases equally sensitive to the inhibitors. The intermediates lycopene and gamma-carotene rejected by chemically inhibited enzymes may be readmitted to other cyclases in the wild type but not in heterokaryons containing a carA mutation. We have calculated the fraction of inhibited cyclase under each condition, the affinity constant of each inhibitor for the cyclase, and the probability that a rejected intermediate molecule will be readmitted and further metabolized. The probabilities for lycopene and gamma-carotene are identical and independent of the inhibitor responsible for the rejection. Our calculations suggest that each rejected intermediate molecule is readmitted to the enzyme aggregates two or three times on the average.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.

Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7 alpha- and 15 beta-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent Km calculated to be 4 mM). K+ and Mg2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24 degrees C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide (ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.

The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA". The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.

An investigation of the reaction mechanism of glutathione reductase isolated from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) was conducted. The enzyme showed GSSG concentration-dependent substrate inhibition by NADPH and pH-dependent substrate inhibition by GSSG. At pH 7.5, the kinetic data were consistent with a basic scheme corresponding to the branching mechanism, involving a ping-pong with formation of a dead-end F.NADPH complex and an ordered sequential mechanism. Both pathways have in common the step in which NADPH binds to the free oxidized form (E) of the glutathione reductase. At low concentrations of GSSG the ping-pong mechanism prevails, whereas at high concentrations the ordered mechanism appears to dominate. The data were analyzed on the basis of the limiting ping-pong mechanism with F.NADPH complex formation and of the hybrid mechanism, and the kinetic constants of the model were calculated. The data obtained at acidic pH values do not rule out the possibility that the kinetic model may be more complicated than the basic scheme studied.

Studies on ATP effects on the allosteric kinetics shown by pyruvate kinase from Phycomyces blakesleeanus NRRL 1555 (-) are reported. Phosphoenolpyruvate showed an allosteric ATP-dependent substrate inhibition. The results supported the existence of spatially distinct catalytic binding sites and the inhibitory binding sites for phosphoenolpyruvate, and ATP showed opposite heterotropic effects with respect to these two types of binding site. With respect to Mg2+ ions, ATP caused a negative heterotropic effect. The global inhibitory effect of ATP was in agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman and Changeux.

The Phycomyces developmental mutant S356 elaborates spores which show a much poorer viability and a higher affinity for Calcofluor White than the wild-type spores. Protease-activated extracts of the mutant spores showed higher levels of chitin synthetase activity than the parental strain-derived spores. High levels of enzyme activity in the mutant extracts, but not in the corresponding wild-type extracts, could be detected in the absence of an exogenous protease. The high basal active chitin synthetase is not the result of activation by endogeneous proteases during cell breakage since protease inhibitors did not reduce, but rather increased, the activity levels. The analysis of cell wall composition in the mutant spores revealed significant changes in the proportion of uronic acids and protein but not in chitin. The mutant phenotype is discussed in relation to the developmental stage at which the alterations connected with cell wall metabolism occurred.

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.