Reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation, and mutagenesis.
Reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation, and mutagenesis.
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DOI：
10.1021/cb500067z
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发表时间：
2014-04-18
影响因子：
4
通讯作者：
Berry, Alan
中科院分区：
文献类型：
--
作者：
Daniels, Adam D.;Campeotto, Ivan;van der Kamp, Marc W.;Bolt, Amanda H.;Trinh, Chi H.;Phillips, Simon E. V.;Pearson, Arwen R.;Nelson, Adam;Mulholland, Adrian J.;Berry, Alan
n -乙酰神经氨酸裂解酶（NAL）是一类醛缩酶，它催化丙酮酸与n -乙酰-d-甘露胺（ManNAc）可逆缩合生成唾液酸n -乙酰神经氨酸（Neu5Ac）。醛缩酶越来越多地被用作复杂分子立体特异性合成的生物催化剂。然而，对醛缩酶催化机制的不完全了解可能会阻碍新酶活性和特异性的发展，包括对新产生的立体中心的控制。在NAL的情况下，很明显，酶催化一个双单有序缩合反应，其中丙酮酸首先与酶结合，形成一个催化重要的希夫碱。然而，催化缩合步骤所需的残留物的身份和该反应的过渡态的性质一直是一个猜测问题。为了解决这个问题，我们在Neu5Ac存在的情况下结晶了大肠杆菌NAL的Y137A变体。三维结构显示全长唾液酸结合在亚基a、B和D的活性位点上，而在亚基C中，不连续的电子密度揭示了酶结合的丙酮酸和甘露聚糖的位置。这些“快照”结构代表了酶催化循环中的中间体，为碳-碳键形成酶反应的QM/MM建模提供了理想的起点。这表明，在反应过程中，Tyr137作为ManNAc醛氧的质子供体，活化屏障以碳-碳键形成为主，需要Tyr137的质子转移才能获得稳定的Neu5Ac-Lys165希夫碱配合物。结果还表明，来自邻近亚基的三个残基Tyr137、Ser47和Tyr110需要正确定位Tyr137才能发挥其功能，这一点通过位点定向突变得到了证实。这种对C-C键形成途径过渡态的机制和几何结构的理解将有助于进一步开发这些酶用于立体定向合成新酶产物。
N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C–C bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.4
作者：
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影响因子：
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作者：
Dobson, Renwick C. J.;Perugini, Matthew A.;Gerrard, Juliet A.
通讯作者：
Gerrard, Juliet A.