L-ornithine decarboxylase from Hafnia alvei has a novel L-ornithine oxidase activity.

Abstract

A novel activity producing gamma-aminobutyric acid (GABA) from L-ornithine in the presence of NAD(P)+ was found in the crude extract of L-ornithine-induced Hafnia alvei, in addition to L-ornithine decarboxylase (ODC) activity. The reaction system for the former activity consisted of two enzymes, L-ornithine oxidase (decarboxylating, OOD) and gamma-aminobutyraldehyde (GABL) dehydrogenase (GDH). OOD catalyzed the conversion of L-ornithine into GABL, CO2, NH3, and H2O2 in the presence of O2, and GDH dehydrogenated GABL to GABA in the presence of NAD(P)+. OOD, purified to homogeneity, had a high ODC activity and the activity ratio of ODC to OOD was almost constant throughout the purification (ODC/ OOD=160:1). The molecular mass of the OOD was about 230 kDa, probably consisting of three identical subunits of a 77 kDa peptide, and OOD had an absorption maximum at 420 nm as well as at 278 nm, the specific absorption for an enzyme containing pyridoxal phosphate (PLP). The content of PLP was estimated at about 1 mol per subunit. OOD was specific to L-ornithine, and other L-amino acids and polyamines including putrescine were inert. The enzyme was activated by PLP, but not by pyridoxamine 5'-phosphate, FAD, FMN, or pyrroloquinoline quinone, and it was inactivated by hydrazine, semicarbazide, and hydroxylamine. The holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted with PLP. These properties of OOD were almost the same as those of ODC separately purified to homogeneity from H. alvei. Zn2+ and Cu2+, butanedione, and sodium borohydride inhibited both OOD and ODC in a similar manner. The OOD reaction required O2 and only the ODC reaction proceeded under anaerobic conditions. The substitution of air for oxygen in the reaction vessel and the addition of catalase-H2O, enhanced only the OOD reaction, resulting in an increase of the ratio of OOD/ODC to 1:30 and 1:4.1, respectively. These results suggested that OOD and ODC are identical and that the former is a side reaction of the latter in the presence of O2.