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
Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds , Leeds LS2 9JT, U.K.
ACS Chem Biol. 2014 Apr 18;9(4):1025-32. doi: 10.1021/cb500067z. Epub 2014 Feb 21.
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.
N-乙酰神经氨酸裂解酶（NAL）是一种I类醛缩酶，催化丙酮酸与N-乙酰-D-甘露糖胺（ManNAc）的可逆缩合反应，生成唾液酸N-乙酰神经氨酸（Neu5Ac）。醛缩酶作为复杂分子立体特异性合成的生物催化剂正得到越来越广泛的应用。然而，对醛缩酶催化机制的不完全理解可能会阻碍新酶活性和特异性的开发，包括对新产生的立体中心的控制。就NAL而言，很明显该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对催化至关重要的席夫碱。然而，缩合步骤催化所需的残基身份以及该反应过渡态的性质一直是个推测的问题。为了解决这个问题，我们在Neu5Ac存在的情况下使大肠杆菌NAL的Y137A变体结晶。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，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ManNAc的位置。这些代表酶催化循环中间体的“快照”结构为碳-碳键形成酶促反应的QM/MM建模提供了理想的起点。这表明在反应过程中Tyr137作为质子供体作用于ManNAc的醛基氧，活化能垒主要由碳-碳键形成主导，并且需要从Tyr137进行质子转移以获得稳定的Neu5Ac-Lys165席夫碱复合物。结果还表明，来自相邻亚基的Tyr137、Ser47和Tyr110这三个残基组合是使Tyr137正确定位以发挥其功能所必需的，这通过定点诱变得到了证实。对沿着碳-碳键形成途径的过渡态机制和几何结构的这种理解将有助于进一步开发这些酶用于新酶产物的立体特异性合成。
