Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus
Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus
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吸水链霉菌氨基糖苷7′′-磷酸转移酶-Ia底物识别的结构基础
DOI：
10.1107/s2053230x19011105
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Yajima Shunsuke
中科院分区：
文献类型：
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作者：
Takenoya Mihoko;Shimamura Tatsuro;Yamanaka Ryuji;Adachi Yuya;Ito Shinsaku;Sasaki Yasuyuki;Nakamura Akira;Yajima Shunsuke
湿霉素B （HygB）是一种氨基糖苷类抗生素，在分子生物学实验中被广泛用作试剂。已知有两种激酶通过磷酸化使HygB失活：吸湿链霉菌的氨基糖苷7 " -磷酸转移酶-Ia [APH(7 ")-Ia]和大肠杆菌的氨基糖苷4-磷酸转移酶-Ia [APH(4)-Ia]。它们分别磷酸化HygB分子7 ‘ ’和4 '位的羟基。此前，APH(4)-Ia的晶体结构被报道为HygB和5′-腺苷酸-β，γ-咪胺二磷酸（AMP-PNP）的三元配合物。为了研究APH（7”）-Ia和APH(4)-Ia在底物识别机制上的差异，我们报道了APH（7”）-Ia与HygB络合的晶体结构。APH（7”）-Ia的整体结构与APH(4)-Ia等其他氨基糖苷磷酸转移酶相似，由一个n端叶（N-lobe）和一个c端叶（C-lobe）组成。后者还包括一个核心和一个螺旋结构域。因此，当APH（7”）-Ia和APH(4)-Ia结构重叠在Brenner基序的三个重要的催化保守残基（His， Asp和Asn）上时，这两个结构在全局上是匹配的，而Brenner基序在氨基糖苷磷酸转移酶和真核蛋白激酶中是保守的。另一方面，两种结构中HygB的磷酸化羟基都靠近Asp残基，并且两种结构中的HygB分子方向相反。这些分子由c叶的螺旋结构域控制，这在两种激酶之间表现出结构差异。此外，根据APH（7”）-Ia和APH(4)-Ia的晶体结构，先前报道的耐热突变体中的一些突变残基位于两种酶的相同位置。
Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′-phosphotransferase-Ia [APH(7′′)-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7′′)-Ia and APH(4)-Ia, the crystal structure of APH(7′′)-Ia complexed with HygB is reported. The overall structure of APH(7′′)-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.