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

从生长在丁胺上的黑曲霉菌丝匀浆的可溶性部分中分离到两种不同的铜藜蛋白胺氧化酶(EC 1.4.3.6)，分别为AO-I和AO-II。Ao-I是一个由两个75 kDa亚基组成的二聚体，而Ao-II是一个80 kDa的单体。纯化的AO-I和AO-II分别呈现粉红色(490 Nm)和黄色(420 Nm)。这些酶表现出与抑制剂类似的底物专一性和敏感性。己胺、丁胺、苄胺、酪胺和组胺是较好的底物。这两种酶都被底物类似物、铜络合剂、一些生物碱和羰基试剂抑制。在中性pH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，在碱性条件下，这些最大吸收峰移动到585 nm。合成的模型化合物TOPQ-海因-对-硝基苯肼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-硝基苯肼的吸收光谱和荧光光谱有显著差异。经热裂解酶消化后，用反相高效液相色谱纯化标记的辅因子多肽，经Edman降解后测序，得到典型的含硫喹酮序列ASN-TOPA-Glu-try(针对AO-II)和非辅因子多肽Val-Val-Ile-Glu-Pro(针对AO-I)，该序列含有与Glu的伽玛-羰基相连的托喹酮，通过质谱学鉴定了辅因子结构。Ao-I和Ao-II的N-末端氨基酸序列不同，并且两种酶的肽谱显示出一些不同的模式。一个编码AO-I的基因已经被克隆，第二个编码AO-II的基因正在处理中。

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.