Characterization of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida

Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.