Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases

Abstract

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

Figure 1

4-Epimerization reactions of sugar phosphates by class II aldolases and epimerase. The products of the enzymatic reactions of class II aldolases and epimerase are 4-OH epimer forms. Class II aldolases can catalyze the 4-epimerization for the specific substrates.

Figure 2

Bio-LC diagram and ³¹P-NMR spectra for the reaction of F6P with FbaA. (A) Bio-LC diagrma of T6P biocoversion from F6P by FbaA. Reactions were carried out at 50 ^oC for 3 h in 50 mM Tris-HCl buffer containing 0.5 mM F6P with FbaA. (B) ³¹P-NMR spectra for the reaction of F6P with FbaA. TEP was ued as the internal standard.

Figure 3

(A) Docking model of FbaA with F6P, T6P, and catalytic residues. They represent pink, green, and red colors, respectively. Green dashed lines represent interactions between the substrates and catalytic residues. Grey dashed lines represent interactions with Zn²⁺. (B) Aldol cleavage and 4-epimerization activities of the wild-type and catalytic residue variant FbaAs from E. coli. Asp288 mediate aldol cleavage via iraction with FBP, whereas Glu182 and Asp288 mediate 4-epimerization via interaction with F6P. Data represent the means of three experiments and error bars represent standard dviations.

Figure 4

Proposed mechanism of FbaA for reversible 4-epimerization. Asp288 or Tyr328 deprotonates the C4-OH of F6P or T6P, splitting F6P or T6P into the enediolate of DHA and G3P. The neutral or acidic form of Glu182 on the opposite face protonates or deprotonates the C3 in the enediolate of DHA or DHA, respectively.

Figure 5

Docking models of AraD, FucA, RhaD, and FbaA for 4-epimerization. The models show the formation of hydrogen bonds at a catalytic acid and a base (bold) when docked with the dominant product of 4-epimerization. One residue forms a hydrogen bond with the C4-OH of the ligand while another forms a hydrogen bond with the C3-OH of the ligand (green), which is responsible for ligand deprotonation, respectively. (A) Docking of X5P in the active site of AraD formed by two adjacent subunits. Asp120 and Asp76 are a catalytic acid and a base for 4-epimerization of l-R5P to X5P, respectively. (B) Docking of l-T1P in the active site of FucA formed by two adjacent subunits. Tyr209 and Glu73 are a catalytic acid and a base residue for 4-epimerization of l-F1P to l-T1P. (C) Docking of S1P in the active site of RhaD formed by two adjacent subunits. Thr200’ and Asp26 are a catalytic acid and a base for 4-epimerization of P1P to S1P, respectively. (D) Docking of T6P in the active site of FbaA. Tyr328 and Glu182 are a catalytic acid and a base for 4-epimerization of F6P to T6P, respectively.