Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography QM/MM Simulation and Mutagenesis

摘要

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-boundpyruvate and ManNAc. These ‘snapshot’ structures, representativeof intermediates in the enzyme catalytic cycle, provided an idealstarting point for QM/MM modeling of the enzymic reaction of carbon–carbonbond formation. This revealed that Tyr137 acts as the proton donorto the aldehyde oxygen of ManNAc during the reaction, the activationbarrier is dominated by carbon–carbon bond formation, and protontransfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165Schiff base complex. The results also suggested that a triad of residues,Tyr137, Ser47, and Tyr110 from a neighboring subunit, are requiredto correctly position Tyr137 for its function, and this was confirmedby site-directed mutagenesis. This understanding of the mechanismand geometry of the transition states along the C–C bond-formingpathway will allow further development of these enzymes for stereospecificsynthesis of new enzyme products.