CHARACTERIZATION OF QUINOHEMOPROTEIN AMINE DEHYDROGENASE FROM PSEUDOMONAS-PUTIDA

Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystalli zed from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultra centrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing max ima at 554, 522, 420, and 320 nm in the reduced form and one peak at 4 10 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxi dized form, The oxidized enzyme was specifically reduced by the additi on of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two mo les of heme c were detected per mole of AMDH and the 60-kDa subunit wa s found to be the heme c-carrying subunit. By redox-cycling quinone st aining, a positive reaction band corresponding to the 20-kDa subunit w as detected after developed by SDS-PAGE, but the 20 kDa band was scarc ely stained by conventional protein staining. Only a silver staining m ethod was possible to detect the subunit after the protein was develop ed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 4 55 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 dehydrogena se in many respects. The presence of an azurin-like blue protein was i dentified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during A MDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstitu ted successfully only by AMDH, the blue protein, and the cytoplasmic m embranes of the organism. The function of the 40-kDa subunit is unknow n at the moment. The properties of AMDH were compared with other bacte rial amine dehydrogenases so far reported.