Bacterial metabolism of alpha-pinene: pathway from alpha-pinene oxide to acyclic metabolites in Nocardia sp. strain P18.3

Griffiths, Ellis; Bociek, S M; Harries, P. C.; Jeffcoat, Roger; Sissons, D J; Trudgill, Peter

doi:10.1128/jb.169.11.4972-4979.1987

Cited by 56 publications

(36 citation statements)

References 13 publications

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“…Best, EMBO Workshop, Geneva, 1986), and its involvement as an intermediate in at-pinene oxidation by the Nocardia strain is supported by circumstantial evidence (3). The lyase is specifically induced by growth of the organisms on oa-pinene and is present at high specific activity (10 to 20 U/mg of protein) in cell extracts (3). The product of ring cleavage has been unequivocally identified as the branched-chain unsaturated aldehyde cis-2-methyl-5-isopropylhexa-2,5-dienal (3); in crude cell extract systems, the reaction does not require oxygen, added electron donors, acceptors, or other components.…”

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confidence: 78%

“…The epoxide has been shown to be formed by an NADH-linked a-pinene monooxygenase in Pseudomonas fluorescens (N. C. Floyd, A. Burfield, A. Magalhaes, P. M. Rhodes, and D. J. Best, EMBO Workshop, Geneva, 1986), and its involvement as an intermediate in at-pinene oxidation by the Nocardia strain is supported by circumstantial evidence (3). The lyase is specifically induced by growth of the organisms on oa-pinene and is present at high specific activity (10 to 20 U/mg of protein) in cell extracts (3).…”

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confidence: 79%

“…Nocardia sp. strain P18.3, isolated from woodland soil, was maintained on nutrient agar slants and grown in liquid medium with oapinene supplied in the vapor phase as described in the accompanying paper (3).…”

Section: Methodsmentioning

confidence: 99%

“…strain P18.3 and certain Pseudomonas strains are grown with a-pinene as the sole source of carbon (3). The epoxide has been shown to be formed by an NADH-linked a-pinene monooxygenase in Pseudomonas fluorescens (N. C. Floyd, A. Burfield, A. Magalhaes, P. M. Rhodes, and D. J.…”

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Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3

et al. 1987

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a-Pinene oxide is an intermediate in the degradation of a-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with a-pinene and constituted 6 to 7% of the soluble protein of cell extracts. The apparent molecular weight of the native enzyme was 50,000 by ultracentrifugal analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave two dissimilar subunits with apparent molecular weights of 17,000 and 22,000. Preparation of terpene "solutions." Compounds of low water solubility were used as aqueous or buffered emulsions prepared as previously described (3). Emulsions of a-pinene oxide were routinely prepared in glycine-NaOH buffer (pH 9.0) to prevent spontaneous decomposition of the compound (6, 7).Enzyme units. All enzyme activity is expressed as micromoles of product formed per minute.Assay of a-pinene oxide lyase. The enzyme was assayed spectrophotometrically at 30°C by following the formation of the ring cleavage product 2-methyl-5-isopropylhexa-2,5-dienal at 235 nm. The initial product of ring cleavage is the cis isomer of the aldehyde (3), but assay in glycine-NaOH buffer at pH 9 (used to suppress spontaneous decomposition of the a-pinene oxide) results in a rapid isomerization of the product to the trans isomer. A typical assay contained, in 1 ml, 0.1 mmol of glycine-NaOH buffer (pH 9.0) and protein (0.025 to 0.15 U of activity). After preincubation at 30°C the reaction was initiated by the addition of 1 ,umol of a-pinene oxide. Activities were calculated using the higher 235 of the trans isomer of 2-methyl-5-isopropylhexa-2,5-dienal (13,000).Purification of a-pinene oxide lyase. All procedures were performed at 2 to 4°C. (i) Step 1. Crude cell extract (15 ml) was diluted to a 70-mi volume with 21 mM Na+-K+-phosphate buffer (pH 7.1) and applied to a DEAE-cellulose column (2.5 by 11 cm). Unbound protein was removed by passage of 21 mM phosphate buffer through the column, and bound proteins were then displaced by passage of a linear gradient, constructed from 250 ml of 21 mM phosphate buffer (pH 7.1) and 250 ml of the same buffer containing 0.5 M KCI, through the column. Fractions (5 ml) were collected. Typically, fractions 61

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confidence: 78%

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confidence: 79%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3

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“…a-Pinene-grown cells of Pseudomonas fluorescens (Best et al, 1987) and a Nocardia sp. strain P18.3 (Griffiths et al, 1987) catalyze the cleavage of both rings of a-pinene oxide ( fig. 5), with the formation of a branched-chain unsaturated aldehyde (ds-2-methyl-5-isopropylhexa-2,5-dienal).…”

Section: Microbial Metabolism Of Epoxyalkanesmentioning

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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