Crystal Structure of 4,6-α-Glucanotransferase GtfC-AC from Thermophilic Geobacillus 12AMOR1:Starch Transglycosylation in Non-Permuted GH70 Enzymes

摘要

GtfC-type 4,6-α-glucanotransferase(α-GT)enzymes from Glycoside Hydrolase Family 70(GH70)are of interest for the modification of starch into low-glycemic index food ingredients.Compared to the related GH70 GtfB-type α-GTs,found exclusively in lactic acid bacteria(LAB),GtfCs occur in non-LAB,share low sequence identity,lack circular permutation of the catalytic domain,and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains.Despite these differences,the first crystal structure of a GtfC,GbGtfC-AC from Geobacillus 12AMOR1,and the first one representing a non-permuted GH70 enzyme,reveals high structural similarity in the core domains with most GtfBs,featuring a similar tunneled active site.We propose that GtfC(and related GtfD)enzymes evolved from starch-degrading α-amylases from GH13 by acquiring α-1,6 transglycosylation capabilities,before the events that resulted in circular permutation of the catalytic domain observed in other GH70 enzymes(glucansucrases,GtfB-type a-GTs).AlphaFold modeling and sequence alignments suggest that the GbGtfC structure represents the GtfC subfamily,although it has a so far unique alternating α-1,4/α-1,6 product specificity,likely determined by residues near acceptor binding subsites +1/+2.