Characterization of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida.

Abstract

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.

Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking

“…Cytochrome c 550 has been shown to be the natural electron acceptor for QHNDH (11). A similar QHNDH has been isolated from Pseudomonas putida, but apparently with somewhat different subunit composition and with exogenous electron acceptor specificity for azurin (12).…”

Crystal Structure of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida

“…Quinohemoprotein amine dehydrogenases (QH-AmDH) from Gram-negative bacteria represent a new type in the quinoprotein class of amine-oxidizing enzymes because they contain not only a quinone but also one or two hemes as a redox active group (7,8) providing an opportunity for intramolecular electron transfer. Intermolecular electron transfer from QHAmDH has been demonstrated with the natural electron acceptors azurin for the enzyme from Pseudomonas putida (7) and cytochrome c-550 for the enzyme from Paracoccus denitrificans (9).…”

The Silent Form of Quinohemoprotein Amine Dehydrogenase fromParacoccus denitrificans

“…This spectral change is characteristic of the redox reaction of heme c groups. The absorption spectra obtained at 0.4 and À0:1 V were almost identical to those of the fully oxidized form (prepared by the oxidation with K 3 Fe(CN) 6 ) and the a b A portion of sQH-AmDH was injected on a mobile phase and mixed with an electrochemically regulated mediator solution at each electrode potential into an MCES system. The electrolyzed mixtures were collected and analyzed QH-AmDH activity.…”

“…16) The concentration of the reduced proteins was determined from the absorbance at 552 nm (" ¼ 37:2 mM À1 cm À1 ). The fully oxidized and reduced forms of QH-AmDH and heme 2 were prepared by adding K 3 Fe(CN) 6 and n-butylamine (or Na 2 S 2 O 4 ) respectively. The subunits of QH-AmDH and heme 2 protein were isolated as previously reported.…”

“…Quinohemoprotein amine dehydrogenase (QHAmDH) is induced when Paracoccus denitrifican 4) and Pseudomonas putida 5,6) are grown on media including biological amines as the sole source of carbon, nitrogen, and energy. QH-AmDH is a soluble bacterial enzyme that catalyzes the oxidative deamination of amines to corresponding aldehydes plus ammonia.…”