Enzymatic synthesis of l-fucose from l-fuculose using a fucose isomerase from Raoultella sp. and the biochemical and structural analyses of the enzyme

Abstract

Background

l-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of Raoultella (RdFucI).

Results

RdFucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn²⁺ as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7–α8 loop of RdFucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity.

Conclusions

RdFucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of RdFucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose.