Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases

Abstract

UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.

Fig. 1

The ratio of UDP-GlcNAc:UDP-GalNAc at specific time-points in reactions for different mutants. X-axis represents the retention time in min and the Y-axis is AU. (A) The ratio of UDP-GlcNAc:UDP-GalNAc at 1 h time-point for all the single-point mutants except Ser306Tyr is ~72:28; (B) The ratio of UDP-GlcNAc:UDP-GalNAc at 1 h time-point and overnight reaction for the double-point mutant Ser144Thr/His305Ala and the triple-point mutant Ser144Thr/Arg304Gly/His305Ala, depicted at the top of each trace.

Fig. 2

F_o–F_c electron density map in the region of UDP-GlcNAc and NAD(H) depicted at an absolute electron density of 0.19 e/ų corresponding to 3.0σ cutoff.

Fig. 3

The structural alignment of wild-type WbgU (grey) with the His503Ala mutant (yellow) viewed along the β-sheet (left) and rotated by ~40°(right). The substrate (UDP-GlcNAc) is bound in the C-terminal domain and the cofactor NAD(H) is bound on the carboxy edge of the β-sheet in the N-terminal domain. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)