Insights into Broad-Specificity Starch Modification from the Crystal Structure of Limosilactobacillus Reuteri NCC 2613 4,6-α-Glucanotransferase GtfB

Abstract

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly “open” and thus suitable as broad-specificity starch-converting enzymes.

Mutations in Amino Acid Residues of Limosilactobacillus reuteri 121 GtfB 4,6-α-Glucanotransferase that Affect Reaction and Product Specificity

“…Biochemical characterization of this enzyme revealed that it is a 4,3-α-glucanotransferase (4,3-α-GTase), converting amylose/maltodextrins to a novel α-glucan with alternating (α1 → 3)/(α 1 → 4) linkages and with (α1 → 3,4) branching points. Overall, Lf2970 GtfB 4,3-α-GTase displayed high sequence identity with Lr121 GtfB 4,6-α-GTase, but homology models suggested that it has a more open binding groove, a feature that recently was shown to be present in about one in five of putative GtfB enzymes and was proposed to determine substrate (and product) specificity. , Moreover, Lf2970 GtfB 4,3-α-GTase has unique variations in some of the residues surrounding the substrate-binding groove, particularly in homology motifs I–IV. In GSs and GtfB, mutations in these motifs were shown to affect acceptor substrate binding and linkage specificity. − ,,− …”

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

“…Despite the low sequence similarity, the GbGtfC-ΔC core structure closely resembles that of GtfB-type 4,6-α-GTs. 18,25,26 Yet, PDBeFold analysis of the core domains (A, B, and C) of the GbGtfC-ΔC crystal structure revealed that the closest structural homologues are α-amylases from Alicyclobacillus sp. (PDB: 6GXV) 48 and Geobacillus stearothermophilus (PDB: 4UZU) 49 with Q-scores of 0.46/0.44 and root-mean-square deviations (RMSD) of 1.95/1.88 Å, respectively.…”

“…Residues H417 (motif II) and Y375 (370s loop) belong to the variable set of residues that have been suggested to affect product specificity in GtfB-type α-GTs. 26 Residue Y375 of GbGtfC is close to subsite +2 and may provide an aromatic stacking platform or a hydrogen bond; for the corresponding P968 of Lr121 GtfB, this is not the case. The larger side chain of Y375 also results in a more constrained acceptor binding space in GbGtfC.…”

“…This initial alignment was extended by three extra sets of sequences representing biochemically characterized bacterial enzymes: (a) eight canonical α-amylases from GH13 subfamily 5 (GH13_5); (b) five GH70 glucansucrase sequences; and (c) six GH70 GtfB sequences. 26 The sequences used for the final alignment are shown in Table S2.…”

“…The bootstrap consensus tree was inferred from 1000 bootstrap replicates. 47 For a comparison between acceptor subsite residues in GtfC-and GtfB-type 4,6-α-GTs, the 63 putative GtfC sequences were aligned with a subset of the 283 putative GtfB sequences from Pijning et al; 26 this subset contained 233 sequences with long loops A1 and B (totaling 37−40 residues), likely featuring a tunneled binding groove.…”