Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.     

Modified ortho-cleavage pathway in Alcaligenes eutrophus JMP134 for the degradation of 4-methylcatechol

Abstract 2,4-Dichlorophenoxyacetate-grown cells of Alcaligenes eutrophus JMP134 [1] metabolized 4-methylphenoxyacetate via a modified ortho -cleavage pathway. 4-Carboxymethyl-4-methylbut-2-en-1,4-olide (4-methyl-2-enelactone), 4-carboxymethyl-3-methylbut-2-en-1,4-olide (3-methyl-2-enelactone) and 4-methyl-3-oxoadipate, were identified as intermediates.     

Adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the TOL plasmid pWW0

We identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol). When the purified enzyme from the recombinant Escherichia coli containing the xylL gene was incubated with toluene cis-dihydrodiol in the presence of NAD+, the end products differed depending on the presence of adenosylcobalamin (coenzyme B12). The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor. Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin. Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction. The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation. To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well.     

Isopentenyl-diphosphate isomerase: inactivation of the enzyme with active-site-directed irreversible inhibitors and transition-state analogues

Seven analogues of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2) containing fluorine, epoxy, and ammonium functional groups irreversibly inhibited isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) from the mold Claviceps purpurea. Inactivation kinetics, substrate protection studies, and labeling experiments demonstrated that the analogues interacted stoichiometrically with the active site of the enzyme. Radioactive enzyme-inactivator complexes were stable to extended dialysis and treatment with chaotropic reagents. The complexes resulting from inactivation of isomerase by 3-(fluoromethyl)-3-buten-1-yl diphosphate (3) and 3,4-epoxy-3-methyl-1-butyl diphosphate (4) were also stable to ion-exchange chromatography and gel electrophoresis. Stoichiometric release of fluoride ion occurred during inactivation of isomerase with 3. This observation is consistent with SN2 or SN2' displacement of fluorine by an active-site nucleophile with concomitant covalent attachment of the inactivator to the enzyme. 2-(Dimethylamino)ethyl diphosphate (9) formed a stable noncovalent complex with isomerase with Kdis less than 1.2 x 10(-10) M. The enzyme-inhibitor complex was stable in 6 M urea, but the inhibitor was partially released upon treatment with SDS and 2-mercaptoethanol at 37 degrees C for 1 h. The results indicate that 9 is a transition-state/reactive intermediate analogue where the positively charged ammonium group mimics a tertiary carbocationic species in the enzyme-catalyzed reaction.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Cloning of beta-isopropylmalate dehydrogenase gene from Bacillus coagulans in Escherichia coli and purification and properties of the enzyme

The beta-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate: NAD+ oxidoreductase, EC 1.1.1.85) gene from Baccilus coagulans was cloned and expressed in Escherichia coli C600, using pBR322 as a vector plasmid. The B. coagulans enzyme was purified to a homogeneous state from the E. coli carrying a pBR322 - the B. coaglulans enzyme gene hybrid plasmid. The enzyme consists of two subunits of equal molecular weight (4.4 X 10(4) ). The enzyme activity was stimulated by 0.5 mM Mn2+, Mg2+ and Co2+. The enzyme was strongly inhibited by 0.2 mM p-chloromercuribenzoate and the inhibition was completely recovered by 1 mM dithiothreitol. The B. coagulans enzyme was thermostabilized by 1.5 M NaCl. The B. coagulans enzyme is a composite of alpha-helix, beta-sheet and remainder. The secondary structure of the enzyme was appreciably altered by 0.5 mM MgCl2 and 1.5 M NaCl.     

Coenzyme stimulation of isomerase activity of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin

The 6-lactoyl tetrahydropterin (C1'-keto PH4) isomerase activity of sepiapterin reductase, which was found in our recent work (Katoh and Sueoka (1987) J. Biochem. 101, 275-278) as a novel activity of the enzyme, i.e., the conversion of C1'-keto PH4 to 6-1'-hydroxy-2'-oxopropyl tetrahydropterin (C2'-keto PH4) without coenzymes, could be enhanced by a small amount of NADPH or NADP+. The concentration of NADP+ required for the maximal stimulation was approximately the same as the concentration of the enzyme subunit. When NADP+ was added with the enzyme and C1'-keto PH4 at pH 8.6, the reaction sequence of C1'-keto PH4----C2'-keto PH4----tetrahydrobiopterin (BH4) was observed in the presence of dithioerythritol. These observations suggest that the coenzyme stimulating the isomerase function of sepiapterin reductase may be involved in the two sequential reductions, from pyruvoyl tetrahydropterin to BH4, by causing internal rearrangement of the keto group of the first intermediate, C1'-keto PH4, to form the second one, C2'-keto PH4.     

Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Delta(1)-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Delta(1)-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Delta(1)-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Synthesis of polyhydroxysterols (III): synthesis and structural elucidation of 24-methylenecholest-4-en-3beta,6 alpha-diol

Using stigmasterol as the starting material, 24-methylenecholest-4-en-3beta,6 alpha-diol (2) was synthesized in eight steps in 13% overall yield. The introduction of the sterol side-chain was carried out using (3-methyl-2-oxobutyl)-triphenylarsonium bromide (11) and K(2)CO(3) in a solid-liquid phase-transfer Wittig reaction. Construction of the steroidal nucleus was finished by oxidation of 24-methylenecholest-5-en-3beta-ol (9) with pyridinium chlorochromate (PCC) in dichloromethane at ambient temperature and by reduction of 24-methylenecholest-4-en-3,6-dione (10) with NaBH(4) in the presence of CeCl(3).7H(2)O.     

Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.     

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions.

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.     

Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine

The biotransformation of dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy,17α-methylandrosta-1,4-dien-3-one) in man was studied with the aim to discover long-term metabolites valuable for the antidoping analysis. Having applied a high performance liquid chromatography for the fractionation of urinary extract obtained from the pool of several DHCMT positive urines, about 50 metabolites were found. Most of these metabolites were included in the GC-MS/MS screening method, which was subsequently applied to analyze the post-administration and routine doping control samples. As a result of this study, 6 new long-term metabolites were identified tentatively characterized using GC-MS and GC-MS/MS as 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol (M3), its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methyl-5β-androst-13-en-3α-ol, 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) and its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methylandrosta-4,13-dien-3α-ol. The most long-term metabolite M3 was shown to be superior in the majority of cases to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-1,4,13-trien-3-one and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one.     

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene and the d-xylulokinase (ATP: d-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The d-xylose isomerase gene was separated from the d-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The d-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the d-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first d-xylose isomerase gene that has been isolated and extensively purified from any organism. d-Xylose isomerase, the enzyme product of the d-xylose isomerase gene, is responsible for the conversion of d-xylose to d-xylulose, as well as d-glucose to d-fructose. It is widely believed that yeast cannot ferment d-xylose to ethanol primarily because of the lack of d-xylose isomerase in yeast. d-Xylose isomerase (also known as d-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the d-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting d-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

Steroid-8-ene isomerase that catalyzes isomerization of delta 8- to delta 7-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and Km values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3 beta-ol is six times that observed with cholest-8-en-3 beta-ol, the delta 8 to delta 7 ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3 beta-ol to cholest-8-en-3 beta-ol; at equilibrium the delta 7-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3 beta-ol and cholest-8(14)-en-3 beta-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3 beta-ol. These results are consistent with the earlier suggestion that delta 8(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.     

Cholest-4-En-3-One-Δ1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ¹-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ¹-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ¹-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.     

Purification and characterization of the -xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli -xylose isomerase ( -xylose ketol-isomerase, EC 5.3.1.5) gene and the -xylulokinase (ATP: -xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The -xylose isomerase gene was separated from the -xylulokinase gene by the construction of a new deletion plasmid, pLX7. The -xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the -xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first -xylose isomerase gene that has been isolated and extensively purified from any organism. -Xylose isomerase, the enzyme product of the -xylose isomerase gene, is responsible for the conversion of -xylose to -xylulose, as well as -glucose to -fructose. It is widely believed that yeast cannot ferment -xylose to ethanol primarily because of the lack of -xylose isomerase in yeast. -Xylose isomerase (also known as -glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the -xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting -xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.