Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3.
alpha-Pinene oxide is an intermediate in the degradation of alpha-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 alpha-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. The enzyme was devoid of prosthetic groups, had no cofactor requirement, and had a broad pH activity range, a Km for alpha-pinene oxide of 9 microM, and a turnover number of 15,000. Inhibitors included sulfhydryl reactive compounds, terpene epoxides, and pinane derivatives with substituent groups at carbon 3. A mechanism for the concerted reaction has been proposed in which decyclization is initiated by donation of a proton from the catalytic center to the oxygen of the epoxide with consequent destabilization. In vitro the enzyme was inactivated during catalysis, and a reactive cationic intermediate may be responsible for this phenomenon. The enzyme should be classified as a lyase EC 4.99.-.-.
Griffiths
E T
ET
Department of Biochemistry, University College of Wales, Aberystwyth, Dyfed, Great Britain.
Harries
P C
PC
Jeffcoat
R
R
Trudgill
P W
PW
D000446:Aldehyde-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology*; D013729:Terpenes / Q000494:pharmacology
D046911:Macromolecular Substances; D013729:Terpenes; D000446:Aldehyde-Lyases; C053996:alpha-pinene oxide lyase