来自碱性磷酸酶超家族的磷酸单酯酶和磷酸二酯酶的比较酶学的机制和进化见解,Journal of the American Chemical Society

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来自碱性磷酸酶超家族的磷酸单酯酶和磷酸二酯酶的比较酶学的机制和进化见解
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2016-10-20 , DOI: 10.1021/jacs.6b06186
Fanny Sunden ₁ , Ishraq AlSadhan ₁ , Artem Y. Lyubimov ₂ , Susanne Ressl ₃ , Helen Wiersma-Koch _{1,

4} , Jamar Borland ₁ , Clayton L. Brown ₁ , Tory A. Johnson ₁ , Zorawar Singh ₁ , Daniel Herschlag _{1,

5}

Affiliation

天真的人可能已经预料到碱性磷酸酶 (AP) 超家族的磷酸单酯酶和二酯酶之间的早期分裂。相反，先前的结果以及我们对来自脑膜败血杆菌的磷酸单酯酶 PafA 的结构和生化分析表明与超家族磷酸二酯酶 [柠檬黄单胞菌核苷酸焦磷酸酶/磷酸二酯酶 (NPP)] 的相似性以及与三种金属离子 AP 超家族单酯酶的明显差异, 来自大肠杆菌 AP (EcAP)。我们进行了一系列实验来绘制和学习这些酶之间的异同。首先，我们问为什么 AP 超家族中会有单酯酶的独立实例？相对于 EcAP，PafA 具有更弱的产物抑制和略高的活性，表明不同的代谢进化压力有利于不同的活性位点结构。接下来，我们分别讨论了 PafA 和 NPP 的优先磷酸单酯和二酯催化。我们询问了这些支架中 >80% 的序列差异是否为每种酶的同源反应提供了功能特化。与该模型的预期相反，PafA 和 NPP 突变体在每个天然支架中嵌入了共同的活性位点组子集，具有相同的单酯酶：二酯酶特异性；因此，天然特异性的 >107 倍差异似乎是由单个磷酰基取代基的不同相互作用引起的。我们还发现了 PafA 和 EcAP 单酯酶之间惊人的机制相似性，包括基态不稳定和功能性活性位点网络的证据，这些网络涉及不同的活性位点组，但可能发挥类似的催化作用。发现常见的网络功能可能会揭示对功能至关重要的活性位点结构连接，并且识别功能模块性区域可能有助于从现有的混杂模板中设计新的酶。更一般地说，比较酶学和催化滥交的分析可以提供机械和进化的见解。识别功能模块区域可能有助于从现有的混杂模板设计新酶。更一般地说，比较酶学和催化滥交的分析可以提供机械和进化的见解。识别功能模块区域可能有助于从现有的混杂模板设计新酶。更一般地说，比较酶学和催化滥交的分析可以提供机械和进化的见解。

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Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily

Naively one might have expected an early division between phosphate monoesterases and diesterases of the alkaline phosphatase (AP) superfamily. On the contrary, prior results and our structural and biochemical analyses of phosphate monoesterase PafA, from Chryseobacterium meningosepticum, indicate similarities to a superfamily phosphate diesterase [Xanthomonas citri nucleotide pyrophosphatase/phosphodiesterase (NPP)] and distinct differences from the three metal ion AP superfamily monoesterase, from Escherichia coli AP (EcAP). We carried out a series of experiments to map out and learn from the differences and similarities between these enzymes. First, we asked why there would be independent instances of monoesterases in the AP superfamily? PafA has a much weaker product inhibition and slightly higher activity relative to EcAP, suggesting that different metabolic evolutionary pressures favored distinct active-site architectures. Next, we addressed the preferential phosphate monoester and diester catalysis of PafA and NPP, respectively. We asked whether the >80% sequence differences throughout these scaffolds provide functional specialization for each enzyme’s cognate reaction. In contrast to expectations from this model, PafA and NPP mutants with the common subset of active-site groups embedded in each native scaffold had the same monoesterase:diesterase specificities; thus, the >107-fold difference in native specificities appears to arise from distinct interactions at a single phosphoryl substituent. We also uncovered striking mechanistic similarities between the PafA and EcAP monoesterases, including evidence for ground-state destabilization and functional active-site networks that involve different active-site groups but may play analogous catalytic roles. Discovering common network functions may reveal active-site architectural connections that are critical for function, and identifying regions of functional modularity may facilitate the design of new enzymes from existing promiscuous templates. More generally, comparative enzymology and analysis of catalytic promiscuity can provide mechanistic and evolutionary insights.

更新日期：2016-10-20

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