Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius

A membrane-bound enzyme activity from Bacillus acidocaldarius converted squalene to two pentacyclic triterpenes, hop-22(29)-ene and hopan-22-ol. The products were formed in a constant molar ratio of hopene:hopanol, 5:1, probably through parallel, and not successive, reactions. The conversion was independent of oxygen, in contrast to the biosynthesis of sterols from epoxysqualene in eukaryotes. The squalene-hopene cyclase was pufified 270-fold by extraction from B. acidocaldarius membranes at low concentrations of Triton X-100 followed by DEAE-cellulose chromatography. The enzyme showed optimal rates of squalene conversion at pH 6 and 60°C, corresponding to the intracellular pH and the optimal growth temperature of the bacterium. The apparent K_m for squalene is 3 μM. Effective inhibitors of the enzyme were some sulfhydryl reagents and the histidyl reagent diethyl pyrocarbonate. The squalene-hopene cyclase, like several eukaryotic epoxysqualene cyclases, was strongly inhibited by AMO 1618 and by high ionic strength. On the basis of these and other similarities a phylogenitic relationship between the dey enzyme of steroid and hopanoid biosynthesis was envisaged.     

Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.

A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K_i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

从酿酒酵母中分离纯化谷胱甘肽

为了提高酵母发酵生产谷胱甘肽的提取率,采用热水直接抽提的方法,并通过正交实验优化及单因素实验优化,得到优化条件：鲜酵母的质量与去离子水体积的比例为1：12;抽提温度90℃;搅拌转速350r／min;当抽提液温度达到90℃就停止抽提,并进行了热水抽提的放大实验。同时在抽提时加氮气保护,防止GSH部分氧化。抽提液离心除菌泥,经截流相对分子质量为10^4的超滤膜超滤后,除去大量杂蛋白,便于后续GSH的精制分离。通过对饱和操作吸附容量及解吸得率的研究,确定强酸型阳离子交换树脂D061为分离介质。     

The purification and characterization of arginase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.     

Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.     

Characterization of NADH kinase from Saccharomyces cerevisiae

At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1,041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105 microM and 2.1 mM, respectively. The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.     

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.     

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.     

Partial purification and some properties of lipoxygenase from Saccharomyces cerevisiae

A partially purified lipoxygenase extract was obtained from the yeast Saccharomyces cerevisiae by precipitation with solid (NH₄)SO₄ at 20% to 80% saturation. The enzyme had two pH optima, at pH 8.0 and 10.0, with respective apparent K _m values of 13 and 9.5 m. At both pH optima, the lipoxygenase demonstrated highest substrate specificity towards linoleic acid, followed by linolenic acid; although the enzyme had less specificity towards mono-linolein than di-linolein at pH 8.0, the reverse was true at pH 10.0.     

Partial purification and characterization of succinyl-CoA synthetase from Saccharomyces cerevisiae

Succinyl-CoA synthetase from Saccharomyces cerevisiae was partially purified (20-fold) with a yield of 44%. The Michaelis-Menten constants were determined: Km (succinate) = 17 mM; Km (ATP) = 0.13 mM; Km (CoA) = 0.03 mM. The succinyl-CoA synthetase has a molecular weight of about 80000 dalton (as determined by polyacrylamide gradient gel electrophoresis). The pH optimum is at 6.0. During fermentation the activity of succinyl-CoA synthetase is lower than in aerobically grown yeast cells. The presence of succinyl-CoA synthetase in fermenting yeasts may be regarded as an indication for the oxidative formation of succinate. In fermenting yeast cells succinyl-CoA synthetase is repressed by glucose if ammonium sulphate serves as nitrogen source. This catabolite repression is not observed with disaccharides or when amino acids are used as nitrogen source.     

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.     

Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants.

Ubiquinol (QH2) is a lipid-soluble molecule that participates in cellular redox reactions. Previous studies have shown that yeast mutants lacking QH2 are hypersensitive to treatment with polyunsaturated fatty acids (PUFAs) indicating that QH2 can function as an antioxidant in vivo. In this study the effect of 1 mM linolenic acid on levels of Q6 and Q6H2 is assessed in both wild-type and respiration-deficient (atp2 delta) strains. The response of Q-deficient mutants to other forms of oxidative stress is further characterized to define those conditions where QH2 acts as an antioxidant. Endogenous antioxidant defense systems were also assessed in wild-type, Q-deficient, and atp2 delta strains. Superoxide dismutase (SOD) activity decreased and catalase activity increased in both Q-deficient and atp2 delta mutants compared to wild-type cells, suggesting that such changes result from the loss of respiration rather than the lack of Q.     

Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I

The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme. The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme. This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation. The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography. The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS-PAGE, and 89 kDa by gel filtration. The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein. Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme.     

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.