Substrate-binding mechanism in trehalose producing enzymes elucidated by crystallography

黒木 良太

no journal, , 

Protein crystallography is one of the powerful tools to clarify substrate-enzyme interactions. It is sometimes problematic to prepare substrate-enzyme complex crystals because of the multiple binding forms of oligo saccharide at the substrate binding site of glycosidases. Maltoolygosyl trehalose trehalohydrolase (MTHase) and maltoolygosyl trehalose synthase (MTSase) are key enzymes for effective trehalose production, and useful enzymes for observing the saccharide orientation between the cleavage subsite -1 and subsite +1 because they are exo-glucosidases and one end of substrate binding site should therefore be blocked. In order to investigate the substrate recognition scheme of these enzymes, the tertiary structure determination of inactive mutants of both MTHase (E283Q) (FEESE,2000) and MTSase (E269Q) in complex with substrate were attempted by X-ray crystallography. The substrate complex did not show any densities belonging to bound substrate in MTSase. Although the structure determination of substrate complex in MTHase was unsuccessful, MTHase-substrate complex was crystallized isomorphously to that of the wild type, and the structure of saccharide bound to subsites -3 to +2 were elucidated (OKAZAKI 2012). The saccharide bound at subsite -1 distorted towards a half chair form, which is stabilized by several hydrogen bonding interactions surrounding the saccharide. This information may be useful to re-design enzymes with increased substrate specificity to maltosyl trehalose.