Sequence and structure‐based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes

Abstract

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino-acidsequence-based classification of FOS synthesizing enzymes would greatly facilitate the in silico identification of novel catalysts, as large amounts of sequence data lie untapped. The development of a bioinformatics tool to rapidly distinguish between high-level FOSs synthesizing predominantly sucrose hydrolysing enzymes from fungal genomic data is presented. Sequence comparison of functionally characterized enzymes displaying lowand high-level FOS synthesis revealed conserved motifs unique to each group. New light is shed on the sequence context of active site residues in three previously identified conserved motifs. We characterized two enzymes predicted to possess low-and high-level FOS synthesis activities based on their conserved motif sequences. FOS data for the enzymes confirmed our successful prediction of their FOS synthesis capacity. Structural comparison of enzymes displaying low-and high-level FOS synthesis identified steric hindrance between nystose and a long loop region present only in low-level FOS synthesizers. This loop is proposed to limit the synthesis of FOS species with higher degrees of polymerization, a phenomenon observed among enzymes displaying low-level FOS synthesis. Conserved sequence motifs surrounding catalytic residues and a distant structural determinant were identifiers of FOS synthesis capacity and allow for functional annotation of sucrolytic enzymes directly from amino acid sequence. The tool presented may also be useful to study the structure-function relationships of b-fructofuranosidases by identifying mutations present in a group of closely related enzymes displaying similar function.

A Horizontal Gene Transfer Led to the Acquisition of a Fructan Metabolic Pathway in a Gall Midge

“…The GH32A group includes enzymes that have been biochemically characterized as having levanase/inulinase activities, [ 28,29 ] while the GH32B group includes enzymes described as having sucrase activity. [ 30 ] HGT of bacterial GH32 has been previously described in Bomby mori [ 31 ] and in Sphenophorus levis . [ 32 ] This atypical adaptive evolution for the efficient digestion of plant‐specific fructans appears to have occurred in multiple species in a punctuated manner, except for the sucrase (GH32B) HGT in Lepidoptera, which apparently occurred in an early evolutionary lineage of Lepidoptera and subsequently was maintained in multiple lepidopteran species (Figure 1C, and Figure S2, Supporting Information).…”

The fructan syndrome: Evolutionary aspects and common themes among plants and microbes

“…These microbes lack specific levanases. Many different fungi employ various GH32 family hydrolases (Trollope, Wyk, Kotjomela, & Volschenk, ; Van der Nest et al, ); however, there have been no reports on fungal GH68 enzymes. 3D structures of two different fungal inulinases from Aspergillus amawori (PDB ID: 1Y4W) and Aspergillus ficuum ( PDB ID: 3RWK); and invertases / β‐fructofuranosidases from Schwanniomyces occidentalis (PDB ID: 3KF5), Aspergillus kawachii (PDB ID: 5XH9), Saccharomyces cerevisiae (PDB ID: 4EQV), and Xanthophyllomyces dendrorhous (PDB ID: 5ANN) have been obtained so far.…”

Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii

“…11) However, the yeast FFases such as SoF-Fase has also transfructosylation activity, 46) and a mutagenesis study of two underlined residues in the WMNDPNG motif (Trp47 and Asn49 in SoFFase) suggested that the motif is involved in hydrolase/transferase activity. 11,47) In addition, two Trp residues (Trp76 and Trp314 in SoFFase) form the catalytic cleft of yeast FFases along with the Asn and Trp residues ( Fig. 6(A)).…”

“…In contrast, FFases that exhibit a weak ability to produce FOS, such as Aureobasidium pullulans FFase, have been found. 11) A combination of alignment and structural analyses of GH32 enzymes indicated that lowlevel FOS-producing enzymes contain a WMNDPNG motif, while high-level FOS-producing enzymes contain a GQIGDPC motif. 11) Aspergillus kawachii IFO 4308 is a filamentous fungus that is used to brew alcoholic beverages.…”

“…11) A combination of alignment and structural analyses of GH32 enzymes indicated that lowlevel FOS-producing enzymes contain a WMNDPNG motif, while high-level FOS-producing enzymes contain a GQIGDPC motif. 11) Aspergillus kawachii IFO 4308 is a filamentous fungus that is used to brew alcoholic beverages. The whole-genome sequence of A. kawachii IFO 4308 has been determined.…”