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 language | English |
|---|---|
| Pages (from-to) | 499-505 |
| Number of pages | 7 |
| Journal | Biochemical Journal |
| Volume | 319 |
| Issue number | 2 |
| Publication status | Published - 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 journal › Article
}
TY - JOUR
T1 - Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2
AU - Chan Kwo Chion, Chan K N
AU - Leak, David J.
PY - 1996/10/15
Y1 - 1996/10/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029843523&partnerID=8YFLogxK
M3 - Article
VL - 319
SP - 499
EP - 505
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 2
ER -