Biochemical characterization of a GH70 protein from Lactobacillus kunkeei DSM 12361 with two catalytic domains involving branching sucrase activity

Abstract

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic cores (CD1 and CD2), predicted to have glucansucrase and branching sucrase specificity, respectively. GtfZ-CD1 was not characterized biochemically due to its unsuccessful expression. With only sucrose as substrate, GtfZ-CD2 was found to mainly catalyze sucrose hydrolysis and leucrose synthesis. When dextran was available as acceptor substrate, GtfZ-CD2 displayed an efficient transglycosidase activity with sucrose as donor substrate. Kinetic analysis showed that the GtfZ-CD2-catalyzed transglycosylation reaction follows a Ping Pong Bi Bi mechanism, indicating the in-turn binding of donor and acceptor substrates in the active site. Structural characterization of the products revealed that GtfZ-CD2 catalyzes the synthesis of single glucosyl (α1 → 3) linked branches onto dextran, resulting in the production of highly branched comb-like α-glucan products. These (α1 → 3) branches can be formed on adjacent positions, as shown when isomaltotriose was used as acceptor substrate. Homology modeling of the GtfZ-CD1 and GtfZ-CD2 protein structure strongly suggests that amino acid differences in conserved motifs II, III, and IV in the catalytic domain contribute to product specificity. Our present study highlights the ability of beneficial lactic acid bacteria to produce structurally complex α-glucans and provides novel insights into the molecular mechanism of an (α1 → 3) branching sucrase.

Keywords: Branching sucrase; GH70; Glucan; Glucansucrase; Lactobacillus kunkeei.