Alkene monooxygenase from Mycobacterium: a multicomponent enzyme

Hartmans, S.; Weber, F.J.; Somhorst, D.P.M.; Bont, J.A.M. de

doi:10.1099/00221287-137-11-2555

Cited by 45 publications

(32 citation statements)

References 33 publications

(28 reference statements)

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“…The aerobic metabolism depends on oxygenases that catalyze hydroxylation reactions with molecular oxygen as co-substrate (12). In the absence of oxygen, alternative biochemical pathways have been identified for hydrocarbon-mineralizing bacteria.…”

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Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Brodkorb

Gottschall

Marmulla

et al. 2010

Journal of Biological Chemistry

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Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ϳ1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mM DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V max ) were 140 nanokatals mg ؊1 for the linalool dehydratase and 410 nanokatals mg ؊1 for the geraniol isomerase, with apparent K m values of 750 M and 500 M, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2

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confidence: 99%

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Brodkorb

Gottschall

Marmulla

et al. 2010

Journal of Biological Chemistry

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“…The ethene monooxygenase from Mycobacterium strain E3 has been examined in some detail (19) and is a multicomponent enzyme, most likely with a binuclear iron active site similar to those of methane and propene monooxygenases (13,39). The reductase component of the monooxygenase from strain E3 has been purified (55), but the other monooxygenase components have not been characterized.…”

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Epoxyalkane:Coenzyme M Transferase in the Ethene and Vinyl Chloride Biodegradation Pathways of Mycobacterium Strain JS60

2003

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Mycobacterium strains that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and are potentially useful for biocatalysis and bioremediation. The catabolic pathway of alkene assimilation in mycobacteria is not well characterized. It is clear that the initial step is a monooxygenase-mediated epoxidation that produces epoxyethane from ethene and chlorooxirane from VC, but the enzymes involved in subsequent transformation of the epoxides have not been identified. We investigated epoxyethane metabolism in Mycobacterium strain JS60 and discovered a coenzyme M (CoM)-dependent enzyme activity in extracts from VCand ethene-grown cells. PCR amplifications using primers targeted at epoxyalkane:CoM transferase (EaCoMT) genes yielded part of the JS60 EaCoMT gene, which was used to clone an 8.4-kb genomic DNA fragment. The complete EaCoMT gene (etnE) was recovered, along with genes (etnABCD) encoding a fourcomponent monooxygenase and two genes possibly involved in acyl-CoA ester metabolism. Reverse transcription-PCR indicated that the etnE and etnA genes were cotranscribed and inducible by ethene and VC. Heterologous expression of the etnE gene in Mycobacterium smegmatis mc 2 155 using the pMV261 vector gave a recombinant strain capable of transforming epoxyethane, epoxypropane, and chlorooxirane. A metabolite identified by mass spectrometry as 2-hydroxyethyl-CoM was produced from epoxyethane. The results indicate that the EaCoMT and monooxygenase enzymes encoded by a single operon (etnEABCD) catalyze the initial reactions in both the VC and ethene assimilation pathways. CoM-mediated reactions appear to be more widespread in bacteria than was previously believed.

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“…Recently, partial purification of the components of AMO from Mycobacterium E3 was attempted. The system was found to be unstable suffering from a very low activity (0.18-3 nmole/min./mg protein) for the production of 1,2-epoxypropane by a mutant of E3 (Hartmans et al, 1989). Demonstration of a stable cell-free activity in Micrococcus sp.…”

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confidence: 99%

Biocatalysts for production of chiral epoxides

Mahmoudian

Michael

1992

Appl Microbiol Biotechnol

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An extensive screen for microorganisms capable of growth on olefins was carried out. Over 100 bacterial strains (mesophilic, thermophilic) were isolated in pure culture with gaseous or liquid (internal & terminal) olefins as the sole source of carbon and energy. The organisms included Aero coccus, Aeromonas,Alcaligenes, Flavobacterium, Micrococcus, Moraxella, Nocardia, Pseudomonas, Staphylococcus, Streptomyces and Vibrio spp. and a variety of Gram-negative, Gram-positive and Gram-variable rods/coccobacilli not yet identified. Given the success of isolating many different genera of alkene-utilizing bacteria, it would suggest that the ability to utilize olefin hydrocarbons is more widespread in nature than previously recognized.Initial studies involved testing a collection of strains for i) growth rate on the substrate of isolation and ii) evidence of epoxide accumulation from lower gaseous olefins. All of the 18 ethene-and propene-utilizing bacteria tested, stereospecifically formed R-1,2-epoxypropane (ee=90-96%), R -l,2-epoxybutane (ee=90-98%) and trans-(2/?,37?)-epoxybutane (ee=64-88%), as analyzed by chiral complexation glc. A particularly promising strain of Micrococcus sp. M90C was selected to demonstrate: a) Induction of epoxidation activity in carbohydrate-grown cells. b) Optimization of a biotransformation process for production of epoxyethane. c) Involvement of a specific monooxygenase in the formation of chiral epoxides. d) Stereoselectivity of epoxyalkane degradation. e) Substrate specificity toward a range of olefins.Micrococcus sp. M90C stereospecifically formed S-(+)-phenyl glycidyl ether (ee=93%) and /?-(+)-phenyl methyl sulphoxide (ee>98%), as analyzed by chiral-shift NMR and HPLC. The ability to catalyze heteroatom oxygenation as well as epoxidation of a wide range of olefins in high optical and chemical yields, demonstrates its potential as a 'general-purpose' biocatalyst for the production of optically pure oxygenated compounds.Two other promising ethene-utilizing bacteria (M26, M93 A) each catalyzed the stereospecific formation of S-(+)-phenyl glycidyl ether (ee=93%). This has attracted considerable industrial interest and is currently under investigation at ICI for commercial production of optically active./1-blockers. In addition to Micrococcus sp. M90C, the substrate specificities of 6 other organisms were studied in order to probe the active site of the enzymes involved. The pattern of reactivity for the group of 4 ethene-(M26, M90C, M93A, M186) and 2 propene-(M142, M l 56) utilizers differed from that expected with peracids, suggesting a considerable degree of enzymatic control.The nature of the substrate, however, modulated enzymatic epoxidations in Staphylcoccus sp. M97B. CONTENTS

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Alkene monooxygenase from Mycobacterium: a multicomponent enzyme

Cited by 45 publications

References 33 publications

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Epoxyalkane:Coenzyme M Transferase in the Ethene and Vinyl Chloride Biodegradation Pathways of Mycobacterium Strain JS60

Biocatalysts for production of chiral epoxides

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