Characterization of Propane Monooxygenase: Initial Mechanistic Studies (original) (raw)
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Applied and environmental microbiology, 1993
A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 +/- 27 and 321 +/- 16 nmol h mg of protein , respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus tric...
Kinetics of 1,4-Dioxane Biodegradation by Monooxygenase-Expressing Bacteria
Environmental Science & Technology, 2006
1,4-Dioxane is a probable human carcinogen, and an important emerging water contaminant. In this study, the biodegradation of dioxane by 20 bacterial isolates was evaluated, and 13 were found to be capable of transforming dioxane. Dioxane served as a growth substrate for Pseudonocardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5, with yields of 0.09 g protein g dioxane -1 and 0.03 g protein g dioxane -1 , respectively. Cometabolic transformation of dioxane was observed for monooxygenaseexpressing strains that were induced with methane, propane, tetrahydrofuran, or toluene including Methylosinus trichosporium OB3b, Mycobacterium vaccae JOB5, Pseudonocardia K1, Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Burkholderia cepacia G4, and Rhodococcus RR1. Product toxicity resulted in incomplete dioxane degradation for many of the cometabolic reactions. Brief exposure to acetylene, a known monooxygenase inhibitor, prevented oxidation of dioxane in all cases, supporting the hypothesis that monooxygenase enzymes participated in the transformation of dioxane by these strains. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene-2-and toluene-4monooxygenases of G4, KR1 and PKO1 were also capable of cometabolic dioxane transformation. Dioxane oxidation rates measured at 50 mg/L ranged from 0.01 to 0.19 mg hr -1 mg protein -1 for the metabolic processes, 0.1-0.38 mg hr -1 mg protein -1 for cometabolism by the monooxygenaseinduced strains, and 0.17-0.60 mg hr -1 mg protein -1 for the recombinant strains. Dioxane was not degraded by M. trichosporium OB3b expressing particulate methane monooxygenase, Pseudomonas putida mt-2 expressing a toluene side-chain monooxygenase, and Pseudomonas JS150 and Pseudomonas putida F1 expressing toluene-2,3dioxygenases. This is the first study to definitively show the role of monooxygenases in dioxane degradation using several independent lines of evidence and to describe the kinetics of metabolic and cometabolic dioxane degradation.
Applied and Environmental Microbiology, 1982
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 (C 1 to C 8 ), 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 NADH 2 or NADPH 2 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 NADH 2 required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potentia...
Applied and environmental microbiology, 2014
Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc(2)-155. Cells of mc(2)-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc(2)-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc(2)-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was a...
Microbial Oxidation ofGaseous Hydrocarbons: Epoxidation of C2toC4n-Alkenes byMethylotrophic Bacteria
1979
Over 20 new cultures of methane-utilizing microbes, including obligate (types I and II) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL Ml NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.
Biochemistry, 2011
Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In the present study we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H 2 O 2 ) generation. Single turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ~50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to generation of H 2 O 2 in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.
Microbial Enzyme Remediation of Poly-Aromatic Hydrocarbon (PAH’s): A review
2022
Pollution of soil by petroleum hydrocarbon (HC) has continued to draw serious concern due to their recalcitrant nature. The HC pollutant are majorly aliphatic and aromatic complexes of incomplete combustion of waste products from automobiles. This HC pollutant can survive in the soil for long time, causing deleterious effect to plant, animals, and humans. Microbial break down or utilization of HC by bacteria and fungi population within the polluted environment can be achieved through biostimulation or bioaugumentation technology by enzymes embedded in the microbial cells. Density, viscosity, pour-point, and solubility are some of the physicochemical parameters that may influence microbial response to HCs. Lack of nutrients, temperature, pH, oxygen are major factors that slows down HC remediation. The degradation of aliphatics by monooxygenase, attacks the terminal methyl group responsible for primary alcohol formation, which is further broken down, to aldehyde and fatty acid. The degradation of aromatics follows dioxygenase-catalyzed oxidation of arenes in aerobic microbial population to yield Vicinal and Vicinal Cis-dihydrodiols Cis, cis-muconic acid (ortho-cleavage) and 2hydroxymuconic semialdehyde (meta-cleavage) is the final product of enzyme catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) catalyzed degradation of HC in the Tricarboxylic Acid Cycle (TCA). The ability of microbial isolates to produce significant enzymes such as C12O, C23O highlights their future remediation significance.
2003
In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [ 14 C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M r s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.