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

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 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.

Styrene Oxide Isomerase‐Catalyzed Meinwald Rearrangement in Cascade Biotransformations: Synthesis of Chiral and/or Natural Chemicals

“…[1][2][3][4] Conventionally, chemical Meinwald rearrangement reactions are catalyzed by Lewis acids or metal complexes, typically requiring harsh conditions and toxic reagents, while also lacking regioselectivity and stereo-control. [5][6][7][8][9][10][11][12][13] Few enzymes catalyze the isomerization of epoxides, [14][15][16] and amongst them only styrene oxide isomerase (SOI) has been used for synthetic applications. SOI catalyzes the Meinwald rearrangement of aryl epoxides to carbonyl compounds with high selectivity under mild conditions, offering an effective biocatalytic alternative to chemical Meinwald rearrangement.…”