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.

Carbohydrates are complex structures that still challenge analysts today because of their different levels of isomerism, notably the anomerism of the glycosidic bond. It has been shown recently that anomerism is preserved upon gas-phase fragmentation and that high-resolution ion mobility (IMS) can distinguish anomers. However, these concepts have yet to be applied to complex biological products. We have used high-resolution IMS on a cyclic device to characterize the reaction products of Uhgb_MS, a novel mannoside synthase of the GH130 family. We designed a so-called IMS n sequence consisting of (i) separating and isolating specific IMS peaks, (ii) ejecting ions to a pre-array store cell depending on their arrival time, (iii) inducing collisional activation upon reinjection, and (iv) performing multistage IMS analysis of the fragments. First, we applied IMS2 sequences to purely linked α1,2- and β1,2-mannooligosaccharides, which provided us with reference drift times for fragments of known conformation. Then, we performed IMS n analyses of enzymatically produced mannosides and, by comparison with the references, we succeeded in determining the intrachain anomerism of a α1,2-mannotriose and a mix-linked β/α1,2-mannotetraosea first for a crude biological medium. Our results show that the anomerism of glycosides is maintained through multiple stages of collisional fragmentation, and that standalone high-resolution IMS and IMS n can be used to characterize the intrachain anomerism in tri- and tetrasaccharides in a biological medium. This is also the first evidence that a single carbohydrate-active enzyme can synthesize both α- and β-glycosidic linkages.