Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.
Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.
复制标题
对大肠杆菌植酸酶立体特异性的结构基础的洞察。
DOI：
10.3390/ijms23116346
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发表时间：
2022-06-06
影响因子：
5.6
通讯作者：
Hemmings, Andrew M.
中科院分区：
文献类型：
--
作者：
Acquistapace, Isabella M.;Thompson, Emma J.;Kuhn, Imke;Bedford, Mike R.;Brearley, Charles A.;Hemmings, Andrew M.
AppA是组氨酸磷酸酶（HP 2）家族的进化枝2的大肠杆菌周质植酸酶，已被充分表征并成功工程化以用作动物饲料补充剂。AppA是一种1D-6-植酸酶，具有高度立体特异性，但可瞬时积累1D-myo-Ins（2，3，4，5）P4和其他低磷酸化中间体。如果要通过蛋白质工程消除正磷酸盐释放的瓶颈，则需要解释其对植酸的起始位点和随后的裂解的相当严格的偏好。为了帮助解释这种行为，催化质子供体残基在确定AppA立体特异性的作用进行了研究。通过含有催化质子供体D304的活性位点HDT氨基酸序列基序的定点诱变产生四种变体。发现潜在质子供体残基的身份和位置强烈影响立体特异性。虽然野生型酶对1D-6-植酸酶活性具有强烈的偏好，但观察到D304 E变体的立体特异性显著降低，而质子供体较少的突变体（D304 A）显示出排他性的1D-1/3-植酸酶活性。突变体与不可水解底物类似物抑制剂的复合物的高分辨率X射线晶体结构指出D304通过影响特异性口袋A和B的大小和极性在立体特异性中发挥的关键作用。两者合计，这些结果提供了参与的质子供体残基在确定HP 2植酸酶的立体特异性的第一个证据，并准备地面结构知情的工程研究，针对生产的动物饲料酶能够有效和完全脱磷酸化的膳食植酸。
AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.
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影响因子：
4.1
作者：
Bedford, Michael R.;Apajalahti, Juha H.
通讯作者：
Apajalahti, Juha H.
影响因子：
8.8
作者：
Cheng, Yim Ling;Andrew, Deborah J.
通讯作者：
Andrew, Deborah J.
影响因子：
3.2
作者：
BOQUET, PL;MANOIL, C;BECKWITH, J
通讯作者：
BECKWITH, J
影响因子：
3.9
作者：
GREINER, R;KONIETZNY, U;JANY, KD
通讯作者：
JANY, KD
影响因子：
2
作者：
Bedford, Michael R.
通讯作者：
Bedford, Michael R.