Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

Enzymatic evaluation of different Aspergillus strains by biotransformation of cyclic ketones

The potential of different Aspergillus strains in carrying out the biotransformation of cyclic ketones was investigated. All the strains employed showed alcohol dehydrogenase and Baeyer–Villiger monooxygenase activities. trans-2-Methylcyclohexanol and trans-4-methylcyclohexanol were prepared in a single isomeric form by the use of Aspergillus terreus SSP 1498 and the corresponding ketones. Baeyer–Villiger oxidation of cyclic ketones by all the fungi Aspergillus led to chiral lactones in good enantioselectivity.     

Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS–PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.     

Regiospecific biotransformation of substituted norbornanones by microorganisms

Summary The regiospecific biotransformation of (±) 5-acetoxy-7-fluoronorbornan-2-one into the equivalent bridgehead lactone of use as a source of substituted cyclopentane synthons containing four successive chiral centres has been demonstrated using washed-cell suspensions of cyclohexanol-grownAcinetobacter sp NCIB 9871. Conditions to optimise production of such 2-oxabicyclooctanones (substrate concentration<25mM, pH 7.35, 30°C) have been characterised.     

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH⁺/CYP2E1⁺) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.     

EPR evidence for nickel-substrate interaction in carbon monoxide dehydrogenase from Clostridium thermoaceticum

Carbon monoxide dehydrogenase from Clostridiumthermoaceticum contains two different Fe₄S₄ rhombic-type EPR resonances with g-values at 2.04, 1.94, 1.90 and 2.01, 1.86, 1.75, respectively. The enzyme after reacting with CO or HCO₃^?/CO₂ also reveals in EPR signal at g = 2.07 and 2.02. This signal, readily observed at 95K, is attributed to a Ni(III) interaction with a radical species formed from CO or HCO₃^?/CO₂.     

Biotransformation of cycloalkenones by fungi Baeyer-Villiger oxidation of bicycloheptenone by dematiaceous fungi

Summary The biotransformation by ring expansion of a bicyclo [3.2.0]-alkenone has been demonstrated in 29 species of dematiaceous fungi. One of these biocatalysts,Curvularia lunata NRRL 2380 has been shown to produce synthetically-important chiral lactones in high yields.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH,NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8–9 U mg^?1 protein (µmoles mg^?1 min^?1) for NADPH and 3–4 U mg^?1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^?1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^?1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^?1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Oxidative biotransformations by microorganisms: production of chiral synthons by cyclopentanone monooxygenase fromPseudomonas sp. NCIMB 9872

Cyclopentanone monooxygenase, an NADPH- plus FAD-dependent enzyme induced by the growth ofPseudomonas sp. NCIMB 9872 on cyclopentanol, has been utilised as a biocatalyst in Baeyer-Villiger oxidations. Washed whole-cell preparations of the microorganism oxidised 3-hexylcyclopentanone in a regio- but not enantioselective manner to give predominantly the racemic γ-hexyl valerolactone. similar preparations biotransformed 5-hexylcyclopent-2-enone exclusively by regio- plus enantioselective oxidation to the equivalent , β-unsaturated (S)-(+)-δ-hexyl valerolactone (ee = 78%), with no reductive biotransformations catalysed by either EC 1.1.x.x- or EC 1.3.x.x-type dehydrogenases.

An equivalent biotransformation of 5-hexylcyclopent-2-enone was catalysed by highly-purified NADPH- plus FAD-dependent cyclopentanone monooxygenase from the bacterium. The regio- plus enantioselective biotransformation by the pure enzyme of 2-(2′-acetoxyethyl)cyclohexanone yielded optically-enriched (S)-(+ )-7-(2′-acetoxyethyl)-2-oxepanone (ee = 72%). The same biotransformation when scaled up again provided optically-enriched (S)-(+)-ε-caprolactone which was converted, using methoxide, to (S)-(−)-methyl 6,8-dihydroxyoctanoate (ee = 42%). thereby providing a two-step access from the substituted cyclohexanone to this important chiron for the subsequent synthesis of (R-(+)-lipoic acid.

Some characteristics of pure NADPH- plus FAD-dependent cyclopentanone monooxygenase were determined including the molecular weight of the monomeric subunit (50000) of this homotetrameric enzyme, and the N-terminal amino acid sequence up to residue 29, which includes a putative flavin nucleotide-binding site.     

Activation of porcine heart mitochondrial malate dehydrogenase by zero valence sulfur and rhodanese.

Incubation of malate dehydrogenase with thiosulfate and rhodanese lead to an increase of dehydrogenasic activity. Selenosulfate, elemental sulfur and elemental selenium were shown similarly able to activate this protein. The activation is limited to the presence of SH groups on the protein. Experiments with ³⁵S demonstrated the direct transfer of zero valence sulfur from rhodanese to malate dehydrogenase. It is proposed that this activation could be a mechanism of enzyme activity modulation in vivo.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

4,4'-Bis dimethylaminodiphenylcarbinol: a new reagent for selective chemical modification. Interaction with porcine malate dehydrogenase

4,4-bis Dimethylaminodiphenylcarbinol (BDC-OH) has recently been reported to be a highly sensitive reagent for the quantitative determination of sulfhydryl residues in biological materials (1). In this communication the effectiveness of BDC-OH as a reagent for selective chemical modification of “active center” cysteine residues was investigated. The supernatant and mitochondrial forms of malate dehydrogenase were chosen for investigation by this reagent. Supernatant malate dehydrogenase which has never been found to contain an “active center” cysteine is unaffected by this reagent. Mitochondrial malate dehydrogenase (L malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart can be irreversibly inactivated by a 20 fold M excess of the reagent. Chemical modification of two essential sulfhydryl residues is prevented by the presence of the coenzyme, NAD⁺, suggesting that the site of interaction is located at or near the coenzyme binding site and hence at or near the enzymatic center of this enzyme.     

A plant protein kinase and plant microsomal H+ transport are stimulated by the ether phospholipid platelet-activating factor

ATP-dependent H⁺ transport in microsomes from zucchini hypocotyls is stimulated by the ether lipid 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor = PAF) known as a hormone-like substance from mammals. The stimulation can only be observed when soluble cytosolic proteins are present. A soluble protein mediating the PAF-dependent H⁺ transport and a PAF-stimulated protein kinase are coeluted by DEAE-Sephacel chromatography. Stimulation of phosphorylation by PAF of a 55 kDa polypeptide without Ca²⁺ and, additionally, of a 35 kDa polypeptide in the presence of Ca²⁺ is observed in zucchini microsomal membranes. This is evidence for a novel phospholipid-stimulated protein kinase in plants. Phosphorylation of regulatory proteins may be involved in the stimulation of in vitro H⁺ transport by PAF.Abbreviations BTP 1,3-bis (tris(hydroxymethyl)-methylamino) propane - DTT dithiotreitol - EGTA ethylene glycol-bis (ß-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - SDS sodium dodecylsulfate - TCA trichloroacetic acid     

A Simple,Preparative Procedure for N 3-Anisoyluridine and O 6-Diphenylcarbamoylguanosine 2′-O-(Tetrahydropyran-2-YL) Derivatives via The Corresponding 3′,5′-Dibenzoates

Abstract

3′,5′-Di-O-benzoyl-2′-O-(tetrahydropyran-2-yl)uridine and 3′,5′ -di-O-benzoyl-N ²-isobutyryl-2′-O-(tetrahydropyran-2-yl)guanosine are converted into-N ³-anisoyl-2′-O-(tetrahydropyran-2-yl)uridine (less and more polar diastereoisomers in 37% and 42% yields, respectively) and O ⁶-diphenyl carbamoylN ²-isobutyryl-2′-O-(tetrahydropyran-2-yl)- guanosine (less and more polar diastereoisomers in 15% and 59% yields, respectively), respectively, by N ³-anisoylation and O ⁶-diphenylcarbamoylation, followed by 3′,5′-di-O-debenzoylation.     

Characterization of an inducible,membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the K_m for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q₁ as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l^-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.Abbreviations cA component A - cB component B - DBMIB dibromomethylisopropyl-benzoquinone - HEPES hydroxyethylpiperazinethanesulfonic acid - IDA iminodiacetate, HN(CH₂COOH)₂ - IDA-DH iminodiacetate dehydrogenase - INT iodonitrotetrazolium chloride - NTA nitrilotriacetate, N(CH₂COOH)₃ - NTA-MO nitrilotriacetate monooxygenase - PMS phenazine methosulphate - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Suc-DH succinate dehydrogenase     

Identification of drostanolone and 17-methyldrostanolone metabolites produced by cryopreserved human hepatocytes

Methyldrostanolone (2α,17α-dimethyl-17β-hydroxy-5α-androstan-3-one) was synthesized from drostanolone (17β-hydroxy-2α-methyl-5α-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3α- (major) and 3β-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3α-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d₀ and TMS-d₉) derivatives. Finally, on the basis of the GC-MS and ¹H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.     

L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

The screening of microorganisms for hydroxylation activity: Hydroxylation of diamantanols

Summary 1-and/or 4-diamantanols are hydroxylated by various species of the genus Rhizopus giving rise to diamantandiols. The GLC method revealed both the species specificity of the organism and substrate specificity in the bioconversion. The effectivity of biotransformation by some species of the genus Rhizopus indicates that the procedure could be used for the preparation of some diamantandiols substituted on tertiary carbon atoms.     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.