Initial Characterization of the Enzyme and Cloning of Genes Involved in the Enantioselective Epoxyalkane Degradation by <i>Xanthobacter</i> PY2

Swaving, Jelto; Weijers, C.A.G.M.; Chion, C. K. Chan Kwo; Leak, David J.; Ooyen, A.J.J. van; Bont, J.A.M. de

doi:10.3109/10242429409065232

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“…150 ml of LMF. In preliminary studies using a 4 ml assay it was shown that, even when 1 ml of this LMF was added to the assay, the reaction was not saturated [12]. Given the rate of attrition that this implied, it was decided at the outset that : (i) standard assays done during purification would be based on DTT-stimulated epoxide disappearance which could be run under saturating DTT concentrations [2], (ii) the LMF-stimulated activity would be used mainly to authenticate results obtained with DTT, and (iii) LMF-based assays would use a fixed, subsaturating concentration of LMF, sufficient to allow determination of the rate of epoxide disappearance within 30 min.…”

Section: Assays Using Lmfmentioning

confidence: 99%

Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2

Chion

Leak

1996

Biochemical Journal

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Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography. Both components were required for the degradation of epoxypropane and were purified further. Component A was apparently homohexameric with a subunit M(r) of about 44,000, and possessed NAD(+)-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity. It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide(NEM)and p-chloromercuribenzoate. Component B was homodimeric with a subunit M(r), of 62,170 and contained 2 mol.mol-1 FAD. It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate. The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane. NADPH was found to replace the need for a low-M(r), fraction in epoxypropane degradation assays containing components A and B and NAD+. The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively. A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed.

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Section: Assays Using Lmfmentioning

confidence: 99%