Structural insight into the substrate specificity of Bombyx mori β-fructofuranosidase belonging to the glycoside hydrolase family 32

Highlights

• Bombyx mori β-fructofuranosidase BmSUC1 showed wide substrate specificity.

• The crystal structure of BmSUC1 was determined.

• The structure of BmSUC1 resembled bacterial GH32 β-fructofuranosidases.

• BmSUC1 was competitively inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol.

• BmSUC1 and its orthologs may contribute to the digestion of β-fructofuranosides.

Abstract

Sucrose-hydrolyzing enzymes are largely divided into β-fructofuranosidase and sucrose α-glucosidase. The domestic silkworm Bombyx mori possesses both enzymes, BmSUC1 and BmSUH, belonging to the glycoside hydrolase family 32 (GH32) and GH13, respectively. BmSUC1 was presumed to be acquired by horizontal gene transfer from bacteria based on phylogenetic analysis and related to tolerance to sugar-mimic alkaloids contained in mulberry latex. Here we investigated the substrate specificity of recombinant BmSUC1 that can hydrolyze not only sucrose but also fructooligosaccharides and fructans, and revealed that the enzyme was competitively inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol, one of the alkaloids. Moreover, the crystal structures of BmSUC1 in apo form and complex with sucrose were determined, and the active site pocket was shallow and suitable for shorter substrates but was related to more relaxed substrate specificity than the strict sucrose α-glucosidase BmSUH. Considering together with the distribution of BmSUC1-orthologous genes in many lepidopterans, our results suggest that BmSUC1 contributes to the digestion of fructooligosaccharides and fructans derived from feed plants.

Introduction

Sucrose is a widely distributed disaccharide, which is one of the main products of photosynthesis used as a carbon source by many organisms. This disaccharide is generally hydrolyzed by glycoside hydrolases (GHs) to produce glucose and fructose, which are primary substrates for glycolysis (Reid and Abratt, 2005; Ruan, 2014). GHs acting on sucrose are divided into two types based on their mechanism. One is β-fructofuranosidase (also known as invertase, EC 3.2.1.26), which recognizes a β-fructofuranosyl residue and hydrolyzes sucrose via a covalent fructosyl-enzyme intermediate (Lammens et al., 2009); the other one is sucrose α-glucosidase (also called sucrase, EC 3.2.1.48) recognizes an α-glucopyranosyl residue and hydrolyzes the α-glucosidic linkages of sucrose (Sim et al., 2010). β-Fructofuranosidases, which are mainly found in bacteria, fungi, and plants, belong to GH32 and GH68 families based on their amino acid sequence homology according to the CAZy database (http://www.cazy.org) (Lombard et al., 2014). These families form the clan GH-J and share the five-bladed β-propeller fold of catalytic domains with the same catalytic machinery (Lammens et al., 2009). On the other hand, sucrases are identified as GH13 from bacteria and insects, GH31 from mammals, and GH100 from bacteria and plants.

The domestic silkworm Bombyx mori possesses two sucrose-hydrolyzing enzymes, BmSUC1 and BmSUH, belonging to GH32 and GH13 subfamily 17 (GH13_17), respectively. BmSUC1 is a secreted enzyme expressed in the midgut and silk glands (Daimon et al., 2008); BmSUH is a membrane-associated enzyme expressed in the midgut and is a major sucrose hydrolase in the lepidopteran species (Wang et al., 2015). GH32 proteins are rarely observed in the animal kingdom, and especially, mammals do not possess GH32 enzymes. However, genes encoding GH32 proteins were reportedly found in only the genomes of Lepidoptera and Coleoptera among insects (Daimon et al., 2008; Pedezzi et al., 2014; Zhao et al., 2014). Phylogenetic analyses indicated that their genes were acquired via horizontal gene transfer (HGT) events from bacteria. B. mori possesses two GH32 genes, BmSUC1 and BmSUC2, the latter of which encodes an inactive protein where the catalytic nucleophile residue is mutated (Daimon et al., 2008). Latex of mulberry, which is the sole feed of B. mori, contains high concentrations of sugar-mimic alkaloids such as 1-deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) (Fig. 1), which are glycosidase inhibitors (Konno et al., 2006). These compounds are harmless to B. mori because BmSUC1 is not inhibited by DNJ, and BmSUH is inhibited by DNJ and DAB but less sensitive than GH13_17 sucrose hydrolases from other lepidopterans (Daimon et al., 2008; Wang et al., 2015).

We recently determined the crystal structure of GH13_17 BmSUH and revealed the mechanisms of sucrose-specific hydrolysis and inhibition by DNJ and DAB (Miyazaki and Park, 2020). Both compounds competitively inhibited BmSUH with K_i values in the micromolar level and were bound to its active site in their complex structures. However, the relationship between these enzymes and tolerance to these alkaloids and why B. mori possesses two sucrose-hydrolyzing enzymes remain unclear. In this study, we examined the substrate specificity toward several β-fructofuranosides and crystal structures of the other isozyme BmSUC1. Combined with biochemical examination, the complex structure with sucrose showed the structure–function relationship in BmSUC1. The results provide novel insights into the substrate recognition mechanism and the physiological function of insect β-fructofuranosidases.