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

Abstract

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

Microbial metabolism and transformation of selected monoterpenes

“…We have proposed a mechanism for the decyclization of a-pinene epoxide which is compatible with the observed properties of the enzyme (Griffiths et al 1987b). Donation of a proton from a site in the catalytic centre initiates a series of concerted rearrangements, leading to formation of the cis isomer of the product (Fig.…”

“…It is possible that l-carvone may arise in small amounts as a side-product during cleavage of a-pinene epoxide by the lyase (EC 4.99.-.-) (Griffiths et al 1987b). Spontaneous cleavage of a-pinene epoxide in acid solution leads to the formation of isomers of carveol (Griffiths et al 1987a) and the proposed mechanism of enzymic ring cleavage (Fig.…”

“…The enzyme was purified from Nocardia P18.3 (Griffiths et al 1987b) and shown to constitute about 6% of the soluble cell protein. Sephacryl S-200 chromatography gave an Mr of 40000 Da and ultracentrifugal determination (Yphantis 1964) a value of 50000 Da.…”

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

“…This isomer would be expected as the initial cleavage product on the basis of the reaction mechanism proposed for the epoxide lyase (12). Isomerization of the aldehyde to the trans isomer occurs nonenzymically in the presence of glycine, and to a lesser extent glycylglycine, although the mechanism of catalysis is not understood.…”

Stability and detection of α‐pinene oxide in aqueous culture medium

“…Based on GC‐MSD comparisons with authentic standards, two of these (peaks b and d) were assigned as (−)‐carveol and sobrerol, respectively. Both of these materials have been previously noted as breakdown products of α‐pinene oxide [10]. Other potential products were tested (see Materials and Methods), but at this time, peaks a, c, and e remain unknown.…”