NALase

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Introduction

Aldolase catalyzes the formation or cleavage of carbon-carbon bonds, and examples can be found in many metabolic pathways. The basic aldol reaction involves the condensation of an aldehyde (the aldol acceptor) with a ketone (the aldol donor) to form a new carbon-carbon bond. Generally speaking, aldolases are highly stereoselective in thier catalysis, and can control the configuration of up to two new chiral centers while forming new carbon-carbon bonds. In this process, there is no need for elaborate protecting groups, and at the same time, the stereoselectivity of aldol condensation is controlled, making the aldolase family an important group of enzymes for biocatalysis. At present, many aldolases have been studied and modified in protein engineering and directed evolution research to improve their utility in chemical synthesis.

N-Acetyl-d-neuraminic acid lyase (NALase) (EC 4.1.3.3) is a pyruvate-dependent aldolase, responsible for catalyzing the reversible aldol condensation between N-acetyl-d-mannosamine (ManNAc) and pyruvate to produce N-acetyl-d-neuraminic acid (Neu5Ac). NALase is a member of the class I aldolase family, which follows an ordered Bi-Uni aldol condensation kinetics mechanism (Scheme 1), in which pyruvate binds first as a Schiff base, followed by ManNAc. Aldol addition then produces Schiff base-bound Neu5Ac, which is then hydrolyzed to release Neu5Ac.  

Figure 1. General Reaction Mechanism of NALase (Daniels, A.D. et al.2014)

After years of hard work, crystallographic studies have determined the three-dimensional NALase structure that includes bound substrates and substrate analogs, but they have not revealed the transition process between these low-energy, stable states. It is almost impossible to obtain structural data of high-energy transition states through crystallography, but this information can be obtained through computational modeling, especially the quantum mechanics/molecular mechanics (QM/MM) methods are very prominent in modeling enzyme reactions.

QM/MM Modeling

After in silicomutate Ala137 back to Tyr, the structure bound pyruvate and ManNAc in subunit C was used as the starting point for QM/MM modeling of the reaction mechanism. Initially, the researchers performed QM/MM umbrella sampling molecular dynamics (MD) simulations alongd(CC). ManNAc, pyruvate-Schiff base and the Tyr137 side chains were included in the QM region, five series of umbrella sampling simulations were performed. In all cases, the Tyr137 hydroxyl proton is spontaneously transferred to ManNAc aldehyde oxygen, and Neu5Ac-Lys165 Schiff base is produced in the final structure.

From the free energy profiles obtained from umbrella sampling, the energy of the intermediate is about 4-8 kcal mol^-1 lower than the transition state. The spontaneity of this reaction and the significantly lower free energy intermediates indicate that Tyr137 is the proton donor in the wild-type NALase reaction, which is consistent with previous speculations.

In the simulations, the water molecule often moves away from this position, and the transfer of proton from Tyr137 to ManNAc aldehyde oxygen occurs as before. Therefore, water molecules should not directly participate in the reaction. However, the Schiff base decomposition process between Lys165 and the substrate/product requires water which can also be used to reprotonate Tyr137 phenate. MD trajectories analysis shows that attacking pyruvate carbons is almost always on the si face, and this preference becomes more pronounced as the carbon atoms are close to each other. Therefore, the simulation strongly suggests that the ManNAc binding mode in wild-type NALase is the main contributor to the reported high stereoselectivity.

Figure 2. QM/MM potential energy profiles for the carbon-carbon bond formation between ManNAc and the pyruvate-Lys165 Schiff base, with Tyr137 as proton donor (Daniels, A.D. et al.2014)

Enzyme Mechanism and Mutagenesis

Based on the crystal structure and QM/MM simulation data introduced above, the detailed mechanism of carbon-carbon bond formation between ManNAc and pyruvate can be proposed.

Figure 3. Mechanism of Carbon–Carbon Bond Formation and Protonation during NALase Catalysis As Derived from the Crystal Structure of the Y137A Variant in the Presence of Pyruvate and ManNAc and QM/MM Modelling (Daniels, A.D. et al.2014)

The enamine form of pyruvate, in Schiff base complex with Lys165 and attacks the si face of ManNAc aldehyde to form Neu5Ac. The increased electron density around the (former) ManNAc aldehyde oxygen (due to the tetrahedral conformation that appears around the aldehyde carbon) is stabilized by hydrogen bonds from the hydroxyls of Thr167 and Tyr137. Once the carbon-carbon bond is formed, the (former) ManNAc aldehyde oxygen is protonated by the Tyr137 hydroxyl group. QM/MM energies indicate that this Neu5Ac Schiff base intermediate with a Tyr137 phenate is very stable.

Two variants at Tyr137 (Y137A and Y137F) have a deleterious effect on activity. It has now been confirmed that this is because Tyr137 plays a vital role in the formation of carbon-carbon bonds between ManNAc and pyruvate. The mutagenesis results also confirmed the importance of Thr167 to stabilize the transition state by forming hydrogen bonds to the (former) aldehyde oxygen. In the case of Tyr110', the substitution of alanine at the most distal of the triad from the active site resulted in a significant decrease in kcat/Km (40-fold), while substitution with the bulkier phenylalanine did not cause a significant change in kinetics.