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

Chan K N Chan Kwo Chion, David J. Leak

Research output: Contribution to journalArticle

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Abstract

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 Mr of about 44000, 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 Mr of 62170 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-Mr 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 a 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.

Original languageEnglish
Pages (from-to)499-505
Number of pages7
JournalBiochemical Journal
Volume319
Issue number2
Publication statusPublished - 15 Oct 1996

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2. / Chan Kwo Chion, Chan K N; Leak, David J.

In: Biochemical Journal, Vol. 319, No. 2, 15.10.1996, p. 499-505.

Research output: Contribution to journalArticle

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abstract = "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 Mr of about 44000, 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 Mr of 62170 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-Mr 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 a 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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