Characterization of quinohemoprotein amine dehydrogenase from Pseudomonas putida
Characterization of quinohemoprotein amine dehydrogenase from Pseudomonas putida
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DOI：
10.1271/bbb.62.469
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发表时间：
1998-03-01
影响因子：
1.6
通讯作者：
Matsushita, K
中科院分区：
文献类型：
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作者：
Adachi, O;Kubota, T;Matsushita, K
从在正丁胺培养基上生长的恶臭假单胞菌 IFO 15366 的可溶部分中纯化并结晶出喹啉血红蛋白胺脱氢酶 (AMDH)。 AMDH 在分析超速离心中给出了单一组分，其固有沉降系数为 5.8s。 AMDH 显示了细胞色素 c 的典型吸收光谱，在还原形式中显示最大值在 554、522、420 和 320 nm 处，在氧化形式中在 410 nm 处有一个峰值，在 350 nm 处有一个肩峰，在 530 nm 附近有一个宽山。氧化酶通过添加胺底物而被特异性还原。 AMDH由60、40和20 kDa 3个不同的亚基组成，总分子量为120,000。每摩尔 AMDH 检测到两摩尔血红素 c，并且发现 60-kDa 亚基是携带血红素 c 的亚基。氧化还原循环醌染色，SDS-PAGE显色后检测到对应20kDa亚基的阳性反应条带，而常规蛋白染色几乎没有对20kDa条带进行染色。蛋白质经 SDS-PAGE 显色后，只能通过银染法检测亚基。对硝基苯肼抑制的 AMDH 解离成亚基，20 kDa 亚基在 455 nm 处显示最大吸收，表明 AMDH 中的羰基辅因子与羰基试剂之间形成席夫碱。因此，AMDH在许多方面不同于非血红素醌蛋白甲胺脱氢酶和芳香胺脱氢酶。在纯化 AMDH 时，从相同的恶臭假单胞菌无细胞提取物中鉴定并纯化了天青蛋白样蓝色蛋白的存在。蓝色蛋白在AMDH反应过程中特异性还原，表明蓝色蛋白是胺氧化中的直接电子受体。胺氧化系统仅由AMDH、蓝色蛋白和生物体的细胞质膜成功重建。 40-kDa 亚基的功能目前未知。将 AMDH 的特性与迄今为止报道的其他细菌胺脱氢酶进行了比较。
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.