Molecular Design of Non-Leloir Furanose-Transferring Enzymes from an α-l-Arabinofuranosidase: A Rationale for the Engineering of Evolved Transglycosylases

Abstract

The vast biodiversity of glycoside hydrolases (GHs) constitutes a reservoir of readily-available carbohydrateacting enzymes that employ simple substrates and hold the potential to perform highly stereopecific and regioselective glycosynthetic reactions. However, most GHs preferentially hydrolyze glycosidic bonds and are thus characterized by a hydrolysis/transglycosylation partition in favor of hydrolysis. Unfortunately, current knowledge is insufficient to rationally modify this partition, specifically mutating key molecular determinants to tip the balance towards transglycosylation. In this study, in the absence of precise knowledge concerning the hydrolysis/transglycosylation partition in a hydrolytic GH51 α-L-arabinofuranosidase, we describe how an iterative protein engineering approach has been used to create the first 'non-Leloir' transarabinofuranosylases. In the first step, random mutagenesis yielded a point mutation (R69H) at a position that is highly conserved in clan GH-A. Characterization of R69H revealed that this enzyme displays high transglycosylation activity, but severely reduced (61-fold) activity on pNP-α-L-arabinofuranoside. Upon recombination of R69H with other point mutations selected using semi-rational or in silico approaches, transfer rates close to 100% and transarabinofuranosylation yields of the main (1 2)-linked oligosaccharide product of 80% (vs 11% for the wild-type) were obtained. Combining data presented here with knowledge drawn from the literature, we suggest that the creation of nonLeloir transglycosylases necessarily involves the destabilization of the highly developed transition states that characterize the predominantly hydrolytic exo-acting GHs; this being an efficient way to prevent ubiquitous water molecules from performing the deglycosylation step.

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

“…They hypothesized that the reduced interactions destabilize the transition states of the reaction, which affects hydrolysis more than transglycosylation and thereby improves the ratio of transglycosylation. The same effect has been seen in several other mutational studies (Arab-Jaziri et al 2015; Aronson et al 2006; Bissaro et al 2015a; Feng et al 2005). …”

“…A stronger nucleophile acceptor may not require this acid catalysis. Furthermore, several mutational studies have indicated the involvement of this tyrosine in modulating the preference for transglycosylation (Bissaro et al 2015a; Teze et al 2014).

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“…TG are rare in nature (Bissaro et al 2015b; Larsbrink et al 2012; Luang et al 2013) but have been successfully produced from retaining GH through directed evolution (Bissaro et al 2015a; Feng et al 2005; Kone et al 2009; Teze et al 2014). Nevertheless, to be able to utilize the vast array of GH for creation of TG, we need to understand what governs their propensity for transglycosylation or hydrolysis.…”

A Single Point Mutation Alters the Transglycosylation/Hydrolysis Partition, Significantly Enhancing the Synthetic Capability of an endo-Glycoceramidase

“…Based on previous studies aimed at improving transglycosylation in rGHs, 14,16,17,[50][51][52][53][54] it is well-established that mutations that lower the hydrolytic component of the reaction and others that increase acceptor binding can lead to overall improvements in transglycosylation.…”

“…Interestingly, the key molecular determinants that differentiate TGs from hydrolytic GHs are still unclearly defined, despite considerable efforts to discover these and relevant studies on reversed transglycosylation/hydrolysis (T/H) partition. [12][13][14][15][16][17] The class of GHs (retaining GHs, hereafter referred to as rGHs) mostly holding the potential to perform transglycosylation reactions are those that catalyze the hydrolysis of glycosidic bonds through a double displacement mechanism that results in the retention (in the product) of the configuration of the anomeric carbon originally present in the donor substrate. The reaction trajectory of rGHs is characterized by the formation of a more or less transient glycosylated enzyme reaction intermediate that can be deglycosylated either by a water molecule (hydrolysis), or by another suitable acceptor moiety (transglycosylation), including sugars and alcohols ( Figure 1).…”