The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis

Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor. We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa. The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N. crassa mutant ( erg-3). The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae. The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH.     

Cloning of the sterol C-14 reductase gene of the tomato pathogenic fungusSeptoria lycopersici and its complementation of theerg-3 mutation ofNeurospora crassa

We have cloned theerg-3 gene, which encodes the ergosterol biosynthetic enzyme sterol C-14 reductase, from the tomato pathogenic fungusSeptoria lycopersici. Its nucleotide sequence, reported here, encodes a 512-amino-acid polypeptide with 54% sequence identity to sterol C-14 reductase ofNeurospora crassa. TheSeptoria gene complemented the pisatin-sensitive, tomatine-resistant and female-sterile phenotypes of aNeurospora erg-3 mutant.     

TheNeurospora crassa erg3 gene encodes a protein with sequence homology to both yeast sterol C-14 reductase and chicken lamin B receptor

Theerg3 gene ofNeurospora crassa was sequenced (EMBL accession no. X77955) and found to encode a protein of 490 amino acid residues with significant homology to the yeast sterol biosynthetic enzyme C-14 reductase (39% identity) and also to the C-tenninal region in the sequence reported for the chicken lamin B receptor (41% identity). The possibility that a single protein may possess both lamin B receptor and sterol C-14 reductase functions might account for non-sterol-biosynthetic effects of mutations in sterol biosynthesis genes and of inhibitors of sterol biosynthetic enzymes.     

Sterol content and enzyme defects of nystatin-resistant mutants of Neurospora crassa.

Summary Non-saponifiable cell extracts of wild type and sterol mutants of N. crassa were analysed by means of gas-liquid chromatography. The wild-type contained ergosterol and episterol in a 10:1 ratio. None of the mutants was able to synthesize ergosterol. Three of the mutants carry single recessive gene mutations causing blocks in the terminal steps of ergosterol biosynthesis: erg-1 has an inactive ^{8 7} isomerase, erg-2 has an inactive 24(28) hydrogenase, and erg-4 has an inactive C-24 methyl transferase. Some of the mutants accumulated novel sterols as a result of their enzyme defects. The genes erg-1 and erg-2 were mapped close to inl on the right arm of chromosome V.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

Restoration of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity of a Neurospora mutant by extracts of various chlorate-resistant mutants of Escherichia coli

Acid-treated extracts of Escherichia coli were tested for their ability to restore reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase activity to an extract of a Neurospora nit-1 mutant which produces a defective enzyme. With wild-type E. coli this complementation activity was more readily detected in the cytoplasmic fraction, although the nitrate reductase activity was found primarily in the particulate fraction. chlB mutants of E. coli appeared to have more complementation activity in the cytoplasm than was observed in the wild type, but no activity in the particulate fraction. The other chl mutants had little or no activity in either fraction. These results suggest that chlB mutants can produce a component or cofactor which is missing in the other mutants and in the Neurospora mutant, but cannot transfer it to the nitrate reductase enzyme.     

In vitro mitochondrial complementation in Neurospora crassa

Mitochondrial isoleucine-valine biosynthesis in strain 330a, an iv-1 mutant of Neurospora, is blocked at the dihydroxy acid dehydration step owing to a mutation in the nuclear structural gene for the specific enzyme dihydroxy acid dehydratase. Dehydratase purified from either the soluble or the mitochondrial fraction of wild-type Neurospora, and incubated in vitro with 330a mitochondria, restores valine synthesis from pyruvate-C ¹⁴ to wild type levels. Up to 29% of the restored synthesis could be attributed to the penetration of enzyme into the mitochondria. However, the bulk of the restored synthesis was found to be mediated via the secretion of dihydroxyvaline (DHV) by the mitochondria into the assay milieu, with subsequent enzymatic catalysis of this metabolite to ketovaline occurring outside the organelle. The ketovaline apparently diffuses back into mitochondria for final transamination to valine. This shunting of valine precursors in and out of mitochondria has been demonstrated to be the mechanism whereby two different populations of mitochondria isolated from mutants 330a and 305A (an iv-2 mutant lacking a functional reductoisomerase) can complement each other for the biosynthesis of valine, even when each population is enclosed in a separate dialysis bag. This observation provides the basis for a biochemical understanding of the growth complementation at the organismic level when these two iv-requiring mutants are cultured together in minimal medium.Work supported by grants GM 12323 and 2TO1 GM 00337 from the National Institutes of Health, USPHS, and a grant from the Robert A. Welch Foundation, Houston, Texas.     

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t _0.5∼10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K _0.5∼100 μM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine. Received: 4 August 1999 / Accepted: 14 February 2000     

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.     

Pneumocystis carinii sterol 14α-demethylase activity in Saccharomyces cerevisiae erg11 knockout mutant: sterol biochemistry

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.     

Carrefour Mme. Gras: a wild-isolated Neurospora crassa strain that suppresses meiotic silencing by unpaired DNA and uncovers a novel ascospore stability defect

Ordinarily, RIP-induced erg-3 mutant Neurospora crassa ascospores and their erg(+) siblings do not differ in stability during long-term storage. Consequently, the frequency of RIP-induced erg-3 mutants remains about constant regardless of the time that has elapsed between ascospore harvest and germination. We found, however, that RIP-induced erg-3 mutants were apparently selectively lost with time from among the ascospores stored from a cross with the wild-isolated Carrefour Mme. Gras strain from Haiti. The Haitian strain was also found to exert a dominant suppression of meiotic silencing by unpaired DNA. Similar loss of RIP-induced erg-3 mutant ascospores was seen among the stored ascospores from a subset of crosses heterozygous for the semi-dominant Sad-1 or Sad-2 suppressors of meiotic silencing. Our results suggest that crosses suppressed in meiotic silencing can compromise the stability during storage of ascospores that inherit RIP-induced mutations.     

Polarised complementation at the pyrimidine-3 locus of Neurospora

Summary A re-evaluation of complementation at the pyrimidine-3 locus ofNeurospora suggests the existence of polarity. This apparent polarity is supported by polar complementation of the majority of a number of mutants induced with the acridine ICR-170, and confirmed by the behavior of an allele which is shown by reversion studies to be a frameshift mutant. Supported by Public Health Service Grant AI-01462.     

Molecular and enzymatic characterizations of novel bifunctional 3beta-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana

We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases (3betaHSD/D) involved in sterol synthesis, termed At3betaHSD/D1 and At3betaHSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3betaHSD/D activity, with the At3betaHSD/D1 isoform or with At3betaHSD/D2 isoform containing a C-terminal At3betaHSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3betaHSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3betaHSD/D isoenzymes showed similar substrate specificities that required free 3beta-hydroxyl and C-4-carboxyl groups but were quite tolerant in terms of variations of the sterol nucleus and side chain structures. Data obtained with 4alpha-carboxy-cholest-7-en-3beta-ol and its 3alpha-deuterated analog revealed that 3alpha-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3betaHSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3beta-hydroxy-4beta,14-dimethyl-5alpha-ergosta-9beta,19-cyclo-24(24(1))-en-4alpha-carboxylic acid, consistent with a decrease in 3betaHSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.     

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Summary Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies.To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene.As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.     

Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.     

Requirement for a second sterol biosynthetic mutation for viability of a sterol C-14 demethylation defect in Saccharomyces cerevisiae.

Genetic analysis of a nystatin-resistant sterol mutant (strain JR4) of Saccharomyces cerevisiae defective in C-14 demethylation revealed the presence of a second mutation in 5,6-desaturation. It appeared from complementation tests that a defect in delta 5-desaturase enzyme activity was required for the viability of the C-14 demethylation mutant. Growth studies with a sterol auxotrophic strain indicated that the major sterol of strain JR4, 14 alpha-methyl-ergosta-8,24(28)-dien-3 beta-ol, could satisfy "bulk" membrane requirements but not the second, structurally specific, sterol function that we defined previously (Rodriguez et al., Biochem. Biophys. Res. Commun. 106:435-441, 1982). Leakiness in the sterol mutations in strain JR4 provided a small amount of ergosterol which could satisfy this second function.     

Forward Genetic Screening for Regulators Involved in Cholesterol Synthesis Using Validation-Based Insertional Mutagenesis

Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resisitant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH₂-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.     

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase

Summary A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.