Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.

Abstract

2, 4-Diaminopentanoate dehydrogenase (2, 4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R, 4S)-2, 4-diaminopentanoate (2, 4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2, 4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2, 4-DAP, indicating that the protein is a 2, 4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2, 4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2, 4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2, 4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2, 4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2, 4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2, 5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.