New Insights into the Fructosyltransferase Activity ofSchwanniomyces occidentalisβ-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

Abstract

Schwanniomyces occidentalis ␤-fructofuranosidase (Ffase) releases ␤-fructose from the nonreducing ends of ␤-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat /K m ) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.

Fructo-Oligosaccharide Synthesis by Mutant Versions of Saccharomyces cerevisiae Invertase

“…In this context, a challenging task is finding out the structural determinants of the docking of the fructosyl moiety and its acceptor in a particular orientation, which will finally result in a specific type of linkage. Several studies in which specific residues have been mutated by site-directed mutagenesis have reported changes in the pattern of products recovered (2,20,27), although the conclusions of these studies still do not offer a complete picture of the transfructosylation mechanism. One of the questions that remains unanswered is which residues determine the formation of either ␤(2,1) or ␤(2,6) linkages (34).…”

“…GH32 enzymes typically contain an additional Cterminal ␤-sandwich domain, which is important for maintaining structural stability and may be involved in protein oligomerization in some cases (1,4). Sequence divergences between hydrolases and transferases belonging to the GH32 family have been the basis for the rationale design of mutations aiming to improve the transfructosylating capacity of different invertases (1,2,26,32). The structural basis of donor and acceptor substrate specificities in transfructosylation reactions has also been studied (20,22).…”

“…In the GH32 enzymes, these three residues (including the nucleophilic Asp, the acid-base Glu, and an Asp residue acting as a transition state stabilizer) are located within three conserved sequences, referred to as the WMNDPNG (or ␤-fructosidase), EC, and RDP motifs, respectively (19). Surrounding the catalytic site, a set of hydrophobic residues, sometimes termed as the hydrophobic pocket, is also important for the stable binding of the substrate, through van der Waals contacts and CHstacking between the aromatic side chains and the sugar rings (2,19,22). GH32 enzymes typically contain an additional Cterminal ␤-sandwich domain, which is important for maintaining structural stability and may be involved in protein oligomerization in some cases (1,4).…”

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

“…Nystose data were generated by HPLC from assays performed under glucose-inhibiting conditions. Sucrose consumption under normal and glucose-inhibiting conditions was quantified by Fourier transform mid-infrared spectroscopy (22). Relative inhibition was expressed as the difference in sucrose consumption between the two conditions divided by uninhibited activity.…”

“…An incomplete understanding of the protein sequence motifs or structural features that impart high fructosyltransferase activity to fungal ␤-fructofuranosidases also precludes the reliable design of mutations to improve catalytic activity or relieve product inhibition. Improvements to fructosyltransferase activity of fungal ␤-fructofuranosidases have been achieved by altering amino acids both in the active-site pocket (21,22) and in the noncatalytic ␤-sandwich domain (20), highlighting the complexities and limitations of rational mutation selection in engineering enzyme activity.…”