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.     

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.     

Carbon isotope composition of CH4 from rice paddies in Japan

Carbon isotope ratios (¹³C) for bubble CH₄ in a submerged paddy soil were studied in Yokohama, Japan, throughout a growing period, and its variation was found. Bubble CH₄ collected from other 33 paddy fields in Japan was also measured for its ¹³C and the results agreed with Yokohama. Furthermore, the variation occurred irrespective of the amount and the type of supplied organic substances to the fields (whole rice straw, rice stubble, or compost). The ¹³C value (average value of -55.9 ± 4.24) from these paddy fields was higher than those of the CH₄ emitted from African and North American paddies. The higher value was little affected by their difference in the supplied organic substances. CH₄ oxidation likely occurs for bubble CH₄ in the shallow paddy fields. A rough estimate of the total CH₄ production, using isotope mass balance, showed that 17 to 22% of organic carbon supplied to Japanese paddies transforms to CH₄.     

Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

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     

Three-year measurement of methane emission from an Indonesian paddy field

Yearly and seasonal (rainy and dry seasons) variations of CH₄ emission from a Sumatra paddy field were measured for 3 years. The mean CH₄ emission rates during the growth period were in the range of 16.0–26.1 mg CH₄ m^-2 h^-1 for the chemical fertilizer plots and 23.3–34.9 mg CH₄ m^-2 h^-1 for the plots with rice straw application, respectively. The increase in the amounts of CH₄ emission by rice straw application were from 1.3 to 1.6 times. There was no significant difference in the mean CH₄ emission rates between rainy and dry seasons.Total amounts of CH₄ emitted during the period of rice growth were in the ranges of 29.5–48.2 and 43.0–64.6 g CH₄ m^-2 for the plots applied with chemical fertilizer and those with rice straw application, respectively. Nearly the same amounts of CH₄ were emitted in the first and second half of the growth period, irrespective of rice straw application.