The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

The involvement of cytochromeP-450 monooxygenase system in aflatoxin biosynthesis byAspergillus flavus

Summary Phenobarbital stimulated aflatoxin biosynthesis byAspergillus flavus and this was paralleled by an increase in microsomal NADPH-cytochromeP-450 reductase and cytochromeP-450 activities. Aflatoxin biosynthesis was inhibited by SKF 525-A, metyrapone and cyanide, inhibitors of the cytochromeP-450 monooxygenase system, further suggesting that aflatoxin biosynthesis byA. flavus could be mediated by a cytochromeP-450 monooxygenase enzyme system.     

Involvement of an Inducible Cytochrome P-450 in Precocene II Oxidation by Streptomyces Griseus

Streptomyces griseus oxidizes the insecticide precocene II to its cis- and trans-dihydrodiols and 3-chromenol after growth on an enriched medium containing soybean flour. Oxidation of precocene II is dependent on the level of cytochrome P-450 in this organism. Extracts of cells grown on media lacking soybean flour were devoid of cytochrome P-450 and could not oxidize precocene II. In an in vitro reconstituted system containing NADPH, spinach ferredoxin reductase, spinach ferredoxin and ammonium sulfate fractions enriched in cytochrome P-450, precocene II was oxidized to its dihydrodiols. An aerial mycelium-negative variant of S. griseus (AMY mutant), that was unable to elicit cytochrome P-450 when grown on soybean flour-enriched medium, failed to oxidize precocene II.     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis

Abstract Methylosinus trichosporium OB3b synthesizes a soluble cytoplasmic methane monooxygenase when grown in copper-depleted medium and a membrane-bound particulate methane monooxygenase under copper-replete conditions. The genes encoding the hydroxylase component of soluble methane monooxygenase, carried on a plasmid in Escherichia coli , were insertionally inactivated using a kanamycin cassette and transferred back into M. trichosporium by conjugation. Marker-exchange mutagenesis, via a double homologous recombination event, yielded a soluble methane monooxygenase-negative mutant which grew only on methane using the particulate methane monooxygenase during copper-replete growth conditions, thus proving that the two methane oxidation systems in this methanotroph are genetically distinct.