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首页> 外文期刊>Journal of bacteriology >Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses.
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Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses.

机译：运动发酵单胞菌中的咪唑丙酮醇磷酸氨基转移酶：分子遗传，生化和进化分析。

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hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.
机译：hisH在运动发酵单胞菌（Zymomonas mobilis）中编码咪唑丙酮醇磷酸酯（IAP）氨基转移酶，位于紧接tyrC上游，该基因编码环己二烯基脱氢酶。含有hisH的质粒能够补充缺乏同源氨基转移酶的大肠杆菌组氨酸营养缺陷型。 hisH的DNA测序揭示了一个1,110 bp的开放阅读框，编码40,631 Da的蛋白质。从大肠杆菌中纯化克隆的hisH产物，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计其分子量为40,000Da。由于通过凝胶过滤测定的天然酶的分子量为85,000 Da，因此活性酶必须是同型二聚体。除磷酸组胺醇外，纯化的酶还能够氨基转移芳香族氨基酸和组氨酸。具有广泛的底物特异性的单一蛋白质的存在与通过各种物理处理（例如冻融，温度失活和吡al醛5'-磷酸酯含量的操纵）后不同底物获得的活性的恒定比率一致。纯化的酶不需要添加吡ido醛5'-磷酸，但是通过羟胺处理拆分了酶和辅因子后，证明了对该辅因子的依赖性。动力学数据显示了氨基转移酶预期的经典乒乓机制。磷酸组氨醇，酪氨酸和苯丙氨酸的Km值为0.17、3.39和43.48 mM。 hisH-tyrC周围的基因结构提示操纵子组织。运动发酵单胞菌中的hisH-tyrC簇使人联想到枯草芽孢杆菌中复杂操纵子的hisH-tyrA组件，其中包括色氨酸操纵子和aroE。数据库中可用的所有氨基转移酶序列的多重比对显示，在I类氨基转移酶超家族中，IAP氨基转移酶（Iβ家族）比Iγ家族（例如大鼠酪氨酸氨基转移酶）更接近Iγ家族（例如，大鼠天冬氨酸转氨酶或大肠杆菌AspC）。在活性位点赖氨酸的区域和域间界面的区域中鉴定了区分IAP氨基转移酶家族的签名基序。

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