Determination of cofactor structure and localization of amine oxidase from Aspergillus niger

从生长在丁胺培养基上的均质化尼日尔曲霉菌丝体的可溶性级分中分离出两种不同的铜-醌蛋白胺氧化酶（EC 1.4.3.6），AO-I和AO-II。AO-I是由两个75 kDa亚基组成的二聚体，而AO-II是80 kDa的单体。纯化的AO-I和AO-II分别显示粉红色（490 nm）和黄色（420 nm）。这些酶显示出相当的底物特异性和对抑制剂的敏感性。这两种酶都能被底物类似物、铜螯合剂、某些生物碱和羰基试剂抑制。在中性条件下，对硝基苯肼标记的酶对AO-I和AO-II的最大吸收波长分别为465和440 nm，碱性条件下，最大吸收波长移到585 nm。合成的模型化合物托帕醌海因对硝基苯腙与标记酶的吸收光谱和荧光光谱具有很好的一致性，而PQQ对硝基苯腙的吸收光谱和荧光光谱存在显著差异。经嗜热菌蛋白酶消化后，用反相高效液相色谱法纯化标记的含辅因子的肽段，Edman降解法测序，得到AO-II的典型含托醌序列Asn-topa-Glu-Try和AO-I的非辅因子肽Val-Val-Ile-Glu-Pro，后者含有与Glu的γ-羰基连接的托醌。AO-I和II的N-末端氨基酸序列被发现是不同的，肽图谱显示了两种酶的一些不同的模式。编码AO-I的基因已被克隆，编码AO-II的第二个基因正在加工中。

Two distinct copper-quinoprotein amine oxidases (EC 1.4.3.6) , AO-I and AO-II,were isolated from the soluble fraction of homogenized Aspergillus niger mycelia grown on butylamine medium. AO-I is a dimmer consisting of two 75 kDa subunits, while AO-II is a monomer of 80 kDa. Purified AO-I and AO-II show pink (490 nm) and yellow (420 nm) colors, respectively. The enzymes show comparable substrate specificity and sinsitivity to inhibitors. Hexylamine butylamine, benzylamine, tyramine and histamine are preferred substrates.Both enzymes are inhibited by substrate analogs, copper chelating agents, some alkaloids and carbonyl reagents. Enzymes labeled with p-nitrophenylhydrazine showed different absroption maxima at 465 and 440 nm for AO-I and AO-II,respectively, in neutral pH.These maxima were shifted to 585 nm under alkali condition.. Absorption spectra and fluorescence spectra of synthesized model compound-topaquinone hydantoin p-nitrophenylhydrazone and labeled enzyme were in very good agreement, while PQQ p-nitrophenylhydrazone showed significant difference. After digestion by thermolysin, labeled cofactor containing peptides were purified using reverse phase HPLC and sequneced by Edman degradation, resulting typical topaquinone containing sequence, Asn-topa-Glu-Try, for AO-II and a noncofactor peptide, Val-Val-Ile-Glu-Pro, which contained topaquinone linked to gamma-carbonyl of Glu, for AO-I.Cofactor structures were confirmed by mass spectrometry. N-Terminal amino acid sequences of AO-I and II were found to be different and peptide mapping showed some distinct patterns for both enzymes. A gene encoding AO-I has been cloned already and the second gene for AO-II is now under processing.