Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase

Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme. Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme. The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme. Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme. Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme. The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar. The mutant enzymes were less thermostable than the wild-type enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.     

Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant.

Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.     

A defect in menadione biosynthesis induces global changes in gene expression in Staphylococcus aureus

Both the high-resolution two-dimensional protein gel electrophoresis technique and full-genome DNA microarrays were used for identification of Staphylococcus aureus genes whose expression was changed by a mutation in menD. Because the electron transport chain is interrupted, the mutant should be unable to use oxygen and nitrate as terminal electron acceptors. Consistent with this, a mutation in menD was found to cause a gene expression pattern typically detected under anaerobic conditions in wild-type cells: proteins involved in glycolytic as well as in fermentation pathways were upregulated, whereas tricarboxylic acid (TCA) cycle enzymes were significantly downregulated. Moreover, the expression of genes encoding enzymes for nitrate respiration and the arginine deiminase pathway was strongly increased in the mutant strain. These results indicate that the menD mutant, just as the site-directed S. aureus hemB mutant, generates ATP from glucose or fructose mainly by substrate phosphorylation and might be defective in utilizing a variety of carbon sources, including TCA cycle intermediates and compounds that generate ATP only via electron transport phosphorylation. Of particular interest is that there are also differences in the gene expression patterns between hemB and menD mutants. While some anaerobically active enzymes were present in equal amounts in both strains (Ldh1, SACOL2535), other classically anaerobic enzymes seem to be present in higher amounts either in the hemB mutant (e.g., PflB, Ald1, IlvA1) or in the menD mutant (arc operon). Only genes involved in nitrate respiration and the ald1 operon seem to be additionally regulated by a depletion of oxygen in the hemB and/or menD mutant.     

Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine

Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.     

Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase.

Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase

Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanediol metabolism.

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for the enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Sulfated N-linked oligosaccharides affect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum

Although previous studies have indicated that N-linked oligosaccharides on lysosomal enzymes in Dictyostelium discoideum are extensively phosphorylated and sulfated, the role of these modifications in the sorting and function of these enzymes remains to be determined. We have used radiolabel pulse-chase, subcellular fractionation, and immunofluorescence microscopy to analyze the transport, processing, secretion, and sorting of two lysosomal enzymes in a mutant, HL244, which is almost completely defective in sulfation. [3H]Mannose-labeled N-linked oligosaccharides were released from immunoprecipitated alpha-mannosidase and beta-glucosidase of HL244 by digestion with peptide: N-glycosidase. The size, Man9-10GlcNAc2, and processing of the neutral species were similar to that found in the wild type, but the anionic oligosaccharides were less charged than those from the wild-type enzymes. All of the negative charges on the oligosaccharides for HL244 were due to the presence of 1, 2, or 3 phosphodiesters and not to sulfate esters. The rate of proteolytic processing of precursor forms of alpha-mannosidase and beta-glucosidase to mature forms in HL244 was identical to wild type. The precursor polypeptides in the mutant and the wild type were membrane associated until being processed to mature forms; therefore, sulfated sugars are not essential for this association. Furthermore, the rate of transport of alpha-mannosidase and beta-glucosidase from the endoplasmic reticulum to the Golgi complex was normal in the mutant as determined by the rate at which the newly synthesized proteins became resistant to the enzyme, endo-beta-N-acetylglucosaminidase H. There was no increase in the percentage of newly synthesized mutant precursors which escaped sorting and were secreted, and the intracellularly retained lysosomal enzymes were properly localized to lysosomes as determined by fractionation of cell organelles on Percoll gradients and immunofluorescence microscopy. However, the mutant secreted lysosomally localized mature forms of the enzymes at 2-fold lower rates than wild-type cells during both growth and during starvation conditions that stimulate secretion. Furthermore, the mutant was more resistant to the effects of chloroquine treatment which results in the missorting and oversecretion of lysosomal enzymes. Together, these results suggest that sulfation of N-linked oligosaccharides is not essential for the transport, processing, or sorting of lysosomal enzymes in D. discoideum, but these modified oligosaccharides may function in the secretion of mature forms of the enzymes from lysosomes.     

Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.     

PHENYLBORIC ACID,A CHEMICAL AGENT SIMULATING THE EFFECT OF THE LANCEOLATE GENE IN THE TOMATO

Lanceolate, a spontaneously occurring mutant in the tomato, which has 1 dosage of the mutant gene, causes a change in the shape of the leaf from the odd-pinnately compound leaf characteristic of the normal tomato plant to a simple, much-reduced in size lanceolate leaf. Homozygous mutant plants, which have 2 dosages of the mutant gene, exhibit extreme lanceolate features, producing a simple narrow leaf, similar in shape to the lanceolate leaf but much smaller in size. The mutant allele of the lanceolate gene causes, in addition to the change in leaf form, the increase in activities of the following oxidative enzymes: tyrosinase, laccase, peroxidase and catalase. The lanceolate gene exhibits a dosage effect with respect to these enzymes, homozygous lanceolate showing the highest activity; heterozygous lanceolate, an intermediate; and normal, the lowest activity. Treatment of germinating normal tomato seeds with phenylboric acid simulated the effect of the mutant allele of the lanceolate gene with regard to both the induction of a lanceolate leaf and the increase in the activity of the above 4 oxidative enzymes; phenylboric acid treatment resulted in relatively higher increase in the activities of the oxidative enzymes in normal and heterozygous lanceolate than in homozygous lanceolate. A direct and close relationship existed between the shape of the leaf and the activity of the enzymes during leaf development. It is suggested that the mutant allele of the lanceolate gene, as well as phenylboric acid, induces an increase in activity of the oxidative enzymes and that this increase in enzyme activities brings about the orderly change in form.     

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.     

Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop

Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype enzyme. Kinetic characterization showed that the V(max) values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced, respectively, compared to the wildtype enzyme. The K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.     

Control of meta-cleavage degradation of 4-hydroxyphenylacetate in Pseudomonas putida.

Synthesis of enzymes of the 4-hydroxyphenylacetate meta-cleavage pathway was studied in Pseudomonas putida wild-type strain P23X1 (NCIB 9865) and mutant strains which had either structural or regulatory gene mutations. Induction studies with mutant strains each defective in an enzyme of the pathway showed that 4-hydroxyphenylacetate induced the hydroxylase and that 3,4-dihydroxyphenylacetate induced the 2,3-oxygenase, aldehyde dehydrogenase, isomerase, decarboxylase, and hydratase. This showed that the hydroxylase structural gene does not exist in an operon that contains any other structural gene of this meta pathway. Studies of mutant strains that synthesized constitutively the 2,3-oxygenase and subsequent enzymes suggested that the regulation of synthesis of these enzymes was coincident, and, in such strains, the hydroxylase was inducible only. Observations made with a putative polarity mutant that lacked 2,3-oxygenase activity suggested that the structural genes encoding this enzyme and subsequent enzymes of the pathway exist in the same operon. Studies of a regulatory mutant strain that was defective in the induction of the 2,3-oxygenase and subsequent enzymes suggest that the 2,3-oxygenase operon is under positive control.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.