Department of Biochemistry, Beckman Center, Stanford University , Stanford, California 94305, United States.
Departments of Molecular and Cellular Physiology, Neurology and Neurological Science, Structural Biology, and Photon Science, Howard Hughes Medical Institute, Stanford University , Stanford, California 94305, United States.
J Am Chem Soc. 2016 Nov 2;138(43):14273-14287. doi: 10.1021/jacs.6b06186. Epub 2016 Oct 20.
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 >10-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.
人们可能会天真地认为，磷酸单酯酶和碱性磷酸酶（AP）超家族的二酯酶会很早就分开。但事实相反，先前的结果以及我们对脑膜败血黄杆菌磷酸单酯酶 PafA 的结构和生化分析表明，它与超家族磷酸二酯酶[桔青霉核苷酸焦磷酸酶/磷酸二酯酶（NPP）]有相似之处，而与来自大肠杆菌的 AP（EcAP）的三种金属离子 AP 超家族单酯酶有明显不同。我们进行了一系列实验，以阐明这些酶之间的异同。首先，我们想知道为什么 AP 超家族会有独立的单酯酶呢？与 EcAP 相比，PafA 的产物抑制作用较弱，活性略高，这表明不同的代谢进化压力有利于不同的活性部位结构。接下来，我们研究了 PafA 和 NPP 对磷酸单酯和二酯的优先催化作用。我们想知道这些支架中 80%以上的序列差异是否为每种酶的同源反应提供了功能专业化。与该模型的预期相反，在每个天然支架中嵌入共同的活性部位基团的 PafA 和 NPP 突变体具有相同的单酯酶：二酯酶特异性；因此，天然特异性的 10 多倍差异似乎来自于单一磷取代基的不同相互作用。我们还在 PafA 和 EcAP 单酯酶之间发现了惊人的机制相似性，包括证据表明基态失稳和功能活性部位网络涉及不同的活性部位基团，但可能发挥类似的催化作用。发现共同的网络功能可能揭示对功能至关重要的活性部位结构连接，并确定功能模块化的区域可能有助于从现有的混杂模板设计新的酶。更一般地说，比较酶学和分析催化混杂性可以提供机制和进化方面的见解。
