Purification and characterization of membrane-bound quinoprotein quinate dehydrogenase
Purification and characterization of membrane-bound quinoprotein quinate dehydrogenase
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DOI：
10.1271/bbb.67.2115
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发表时间：
2003-10-01
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;Yoshihara, N;Matsushita, K
通过醋酸菌和其他细菌筛选出了几株携带醌蛋白奎尼酸脱氢酶（QDH）的菌株。在生黑葡糖杆菌IFO 3294、氧化葡糖杆菌IFO 3292、氧化葡糖杆菌IFO 3244以及一些乙酸钙不动杆菌菌株的膜组分中发现了较强的酶活性。有趣的是，在不能产生吡咯并喹啉醌（PQQ）的乙酸钙不动杆菌AC3的膜组分中，形成了大量的脱辅基 - QDH，并且只有添加PQQ时才会转化为全酶 - QDH。通过乙二胺四乙酸（EDTA）处理很难从全酶 - QDH上分离PQQ，并且在添加PQQ之前用EDTA处理脱辅基 - QDH没有产生明显的全酶 - QDH。对于QDH的纯化，葡糖杆菌菌株由于在同一膜中存在大量的醌血红蛋白醇脱氢酶（ADH）而不合适，ADH与QDH共同溶解并干扰QDH的纯化。而是使用不含ADH的不动杆菌属SA1进行全酶 - QDH的纯化。从乙酸钙不动杆菌AC3纯化脱辅基 - QDH。这是第一篇关于QDH纯化的报道，并给出了两种不同的QDH纯化标准。使用丁基 - Toyopearl柱和羟基磷灰石柱的两步组合得到了高度纯化的全酶 - QDH，其呈单分散状态且纯度足够，尽管比活性没有像预期的那样大幅提高。当在没有PQQ的情况下用乙酸钙不动杆菌AC3进行QDH纯化时，纯化的脱辅基 - QDH似乎是一个二聚体，添加PQQ后会转化为单体。由于QDH具有高度疏水性，尝试了在DEAE - Sepharose柱上进行一步色谱法。以更高的产率获得了比活性更高的纯化全酶 - QDH。QDH的分子量估计为88 kDa。除了在420nm左右有一个小峰外，纯化的QDH没有特征吸收光谱。到目前为止所检测的，QDH仅氧化奎尼酸和莽草酸。当用人工电子受体测定时，在pH 6 - 7时发现QDH活性最佳。在培养基中有无奎尼酸的情况下都会形成QDH，尽管在有奎尼酸存在时通常会观察到更强的诱导作用。
Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant bolo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3.This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 run. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.