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

Abstract

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-ΔC 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 α-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.

Unprecedented Diversity of the Glycoside Hydrolase Family 70: A Comprehensive Analysis of Sequence, Structure, and Function

“…reuteri 121 (PDB: 5JBF ) complexed with maltopentaose (orange carbon atoms) which was extended to maltooctaose (G8) by modeling. (d) GtfC-type 4,6-α-glucanotransferase from Geobacillus 12AMOR1 (PDB: 7ZC0 ]) with a modeled maltooctaose.…”

“…Almost all of these were already predicted from the sequences and in earlier published AlphaFold models of GtfC-like α-GTs. 26 First, in 8 of the 10 enzymes, two copies of a type 2 bIG domain (bIG_2; IPR003343; pfam02368) are predicted, displaying a twolayered sandwich of β-sheets in a Greek key motif. Alignment of all bIG_2 domains (Figure S6a) shows a moderate conservation of aromatic residues (Figure S6b) and some exposed on the domain surface.…”

“…12−18 Whether this applies to all GH70 enzymes is so far unknown, but 3D modeling of some GtfC-type α-GTs suggested that other topologies are likely to exist in auxiliary domains. 26 The last decades have seen a growing interest in the αglucan products of GH70 enzymes, which can be synthesized from relatively cheap substrates in biobased, eco-friendly routes. 8,10,11 Due to the promiscuity of GH70 enzymes regarding the acceptor reaction, a wide range of glucosylated sugars or hydroxylated compounds can be synthesized efficiently.…”

“…Similar to GS and BrS, to study α-GTs, their protein sizes have been reduced by using N- and C-terminally truncated constructs that lack significant parts of noncore (auxiliary) domains. Crystal structures have been reported for GtfB-type 4,6-α-GTs from four different Limosilactobacillus species, − as well as for the GtfC-type 4,6-α-GT from Geobacillus 12AMOR1 …”

“…Strategy for creating a representative multiple sequence alignment of GH70 enzymes. (a) Flow scheme used to combine permuted (left) and nonpermuted (right) sequences, respectively represented by glucansucrase Gtf180 from L. reuteri 180 (LrGtf180)12 and 4,6-αglucanotransferase GtfC from Geobacillus 12AMOR1 (GbGtfC) 26. (b) Organization and reordering of domains A, B, C and IV in permuted and nonpermuted sequences to allow their alignment.…”