Azasterol inhibition of delta 24-sterol methyltransferase in Saccharomyces cerevisiae

The inhibition of the delta 24-sterol methyltransferase (24-SMT) of Saccharomyces cerevisiae by side-chain azasterols is related to their nuclear skeleton and side chain nitrogen position. Inhibitory power [I50 (microM)] was found to be in the order of 25-azacholesterol hydrochloride salt (0.05) greater than 25-aza-24,25-dihydrozymosterol (0.08) greater than 25-azacholesterol approximately equal to 25-azacholestanol (0.14) greater than (20R)- and (20S)-22,25-diazacholesterol (0.18) greater than 24-azacholesterol (0.22) greater than 25-aza-24,25-dihydrolanosterol (1.14) greater than 23-azacholesterol (4.8). In the presence of azasterols, S. cerevisiae produces increased amounts of zymosterol, decreased amounts of ergosterol and ergostatetraenol, and the new metabolites cholesta-7,24-dienol, cholesta-5,7,24-trienol, and cholesta-5,7,22,24-tetraenol. Kinetic inhibition studies with partially purified 24-SMT and several azasterols suggest the azasterols act uncompetitively with respect to zymosterol and are competitive inhibitors with respect to S-adenosyl-L-methionine (SAM). These results are consistent with at least two kinetic mechanisms. One excludes competition of azasterol and zymosterol for the same site, whereas a second could involve a ping-pong mechanism in which 24-SMT is methylated by SAM and the methylated enzyme reacts with sterol substrate.     

Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae.

Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine. Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We have shown that yeast MET11 is a functional homologue of human MTHFR. Expression of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrophic phenotype in vivo. To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of the enzyme. However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization. We have also functionally characterized four missense mutations identified from patients with severe MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population. Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme.     

Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity.     

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.     

Sterol 24(28) methylene reductase in Saccharomyces cerevisiae.

Optimal conditions for the 24(28)methylene reductase were obtained. The enzyme assay provided for unusually high activity; the Km was determined to be 10.8 mum. The enzyme activity was increased in cells grown with ethanol as the substrate.     

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.     

Mutational spectrum in the Delta7-sterol reductase gene and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive malformation syndrome, ranges in clinical severity from mild dysmorphism and moderate mental retardation to severe congenital malformation and intrauterine lethality. Mutations in the gene for Delta7-sterol reductase (DHCR7), which catalyzes the final step in cholesterol biosynthesis in the endoplasmic reticulum (ER), cause SLOS. We have determined, in 84 patients with clinically and biochemically characterized SLOS (detection rate 96%), the mutational spectrum in the DHCR7 gene. Forty different SLOS mutations, some frequent, were identified. On the basis of mutation type and expression studies in the HEK293-derived cell line tsA-201, we grouped mutations into four classes: nonsense and splice-site mutations resulting in putative null alleles, missense mutations in the transmembrane domains (TM), mutations in the 4th cytoplasmic loop (4L), and mutations in the C-terminal ER domain (CT). All but one of the tested missense mutations reduced protein stability. Concentrations of the cholesterol precursor 7-dehydrocholesterol and clinical severity scores correlated with mutation classes. The mildest clinical phenotypes were associated with TM and CT mutations, and the most severe types were associated with 0 and 4L mutations. Most homozygotes for null alleles had severe SLOS; one patient had a moderate phenotype. Homozygosity for 0 mutations in DHCR7 appears compatible with life, suggesting that cholesterol may be synthesized in the absence of this enzyme or that exogenous sources of cholesterol can be used.     

Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae.

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.     

Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae.

Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Identification of essential elements in U14 RNA of Saccharomyces cerevisiae.

The U14 RNA of Saccharomyces cerevisiae is a small nucleolar RNA (snoRNA) required for normal production of 18S rRNA. Depletion of U14 results in impaired processing of pre-rRNA, deficiency in 18S-containing intermediates and marked under-accumulation of mature 18S RNA. The present report describes results of functional mapping of U14, by a variety of mutagenic approaches. Special attention was directed at assessing the importance of sequence elements conserved between yeast and mouse U14 as well as other snoRNA species. Functionality was assessed in a test strain containing a galactose dependent U14 gene. The results show portions of three U14 conserved regions to be required for U14 accumulation or function. These regions include bases in: (i) the 5'-proximal box C region, (ii) the 3'-distal box D region, and (iii) a 13 base domain complementary to 18S rRNA. Point and multi-base substitution mutations in the snoRNA conserved box C and box D regions prevent U14 accumulation. Mutations in the essential 18S related domain do not effect U14 levels, but do disrupt synthesis of 18S RNA, indicating that this region is required for function. Taken together, the results suggest that the box C and box D regions influence U14 expression or stability and that U14 function might involve direct interaction with 18S RNA.     

Saccharomyces cerevisiae chorismate synthase has a flavin reductase activity

Chorismate synthase (CS) catalyses the conversion of 5- enol pyruvylshikimate 3-phosphate (EPSP) to form chorismate, which is the last common intermediate in the synthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. Despite the overall redox-neutral reaction, catalysis has an absolute requirement for reduced flavin. In the fungus Neurospora crassa , a flavin reductase (FR) activity able to generate reduced flavin mononucleotide in the presence of NADPH is an intrinsic feature of a bifunctional CS. In all bacterial and plant species investigated to date, purified CSs lack an FR activity and are correspondingly 8–10 kDa smaller than the N. crassa CS (on the basis of SDS–PAGE). The cloning of N. crassa CS and subsequent characterization of the purified heterologously expressed enzyme indicates that, surprisingly, the FR probably resides within a region conserved amongst both mono- and bifunctional CSs and is not related to non-homologous sequences which contribute to the larger molecular mass of the N. crassa CS. This information directed this work towards the smaller Saccharomyces cerevisiae CS, the sequence of which was known, although the protein has not been extensively characterized biochemically. Here the characterization of the S. cerevisiae CS is reported in more detail and it is shown that the protein is also bifunctional. With this knowledge, S. cerevisiae could be used as a genetic system for studying the physiological consequences of bifunctionality. The phylogenetic relationship amongst known CSs is discussed.     

Saccharomyces cerevisiae ACR2 gene encodes an arsenate reductase

The ACR2 gene of Saccharomyces cerevisiae was disrupted by insertion of a HIS3 gene. Cells with the disruption were sensitive to arsenate. This phenotype could be complemented by ACR2 on a plasmid. The ACR2 gene was cloned and expressed in Escherichia coli as a malE gene fusion with a C-terminal histidine tag. The combination of chimeric MBP-Acr2-6H protein and yeast cytosol from an ACR2-disrupted strain exhibited arsenate reductase activity.     

The redox interconversion mechanism of Saccharomyces cerevisiae glutathione reductase

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae

Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.