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 (similar to ninefold) was achieved at 30 degrees 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 degrees C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 degrees 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 alpha 7-alpha 8 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.