Y418 in 410s loop is required for high transglucosylation activity and large-ring cyclodextrin production of amylomaltase from Corynebacterium glutamicum

Biochemical and Biophysical Research Communications
Suthipapun TumhomPiamsook Pongsawasdi

Abstract

Amylomaltase catalyzes α-1,4 glucosyl transfer reaction to yield linear or cyclic oligosaccharide products. The aim of this work is to investigate functional roles of 410s loop unique to amylomaltase from Corynebacterium glutamicum (CgAM). Site-directed mutagenesis of Y418, the residue at the loop tip, was performed. Y418A/S/D/R/W/F - CgAMs were characterized and compared to the wild-type (WT). A significant decrease in starch transglucosylation, disproportionation and cyclization activities was observed. Specificity for G3 substrate in disproportionation reaction was not changed; however, Y418F showed an increase in preference for longer oligosaccharides G5 to G7. The catalytic efficiency of Y418 mutated CgAMs, except for Y418F, was significantly lower (up to 8- and 12- fold for the W and R mutants, respectively) than that of WT. The change was in the kcat, not the Km values which were around 16-20 mM. The profile of large-ring cyclodextrin (LR-CD) product was different; the principal product of Y418A/D/S was shifted to the larger size (CD36-CD40) while that of the WT and Y418F peaked at CD29-CD33. The product yield was reduced especially in W and R mutants. Hence Y418 in 410s loop of CgAM not only contributes to transglucosyl...Continue Reading

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