De novo design of a trans- -N-acetylglucosaminidase activity from a GH1  -glycosidase by mechanism engineering

Abstract

Glycoside hydrolases are particularly abundant in all areas of metabolism as they are involved in the degradation of natural polysaccharides and glycoconjugates. These enzymes are classified into 133 families (CAZy server, http://www.cazy.org) in which members of each family have a similar structure and catalytic mechanism. In order to understand better the structure/function relationships of these enzymes and their evolution and to develop new robust evolved glycosidases, we undertook to convert a Family 1 thermostable β-glycosidase into an exo-β-N-acetylglucosaminidase. This latter activity is totally absent in Family 1, while natural β-hexosaminidases belong to CAZy Families 3, 20 and 84. Using molecular modeling, we first showed that the docking of N-acetyl-d-glucosamine in the subsite -1 of the β-glycosidase from Thermus thermophilus (TtβGly) suggested several steric conflicts with active site amino-acids (N163, E338) induced by the N-acetyl group. Both N163A and N163D-E338G mutations induced significant N-acetylglucosaminidase activity in TtβGly. The double mutant N163D-E338G was also active on the bicyclic oxazoline substrate, suggesting that this mutated enzyme uses a catalytic mechanism involving a substrate-assisted catalysis with a noncovalent oxazoline intermediate, similar to the N-acetylglucosaminidases from Families 20 and 84. Furthermore, a very efficient trans-N-acetylglucosaminidase activity was observed when the double mutant was incubated in the presence of NAG-oxazoline as a donor and N-methyl-O-benzyl-N-(β-d-glucopyranosyl)-hydroxylamine as an acceptor. More generally, this work demonstrates that it is possible to exchange the specificities and catalytic mechanisms with minimal changes between phylogenetically distant protein structures.

Design, Synthesis, and Preliminary Biological Evaluation of GlcNAc‐6P Analogues for the Modulation of Phosphoacetylglucosamine Mutase 1 (AGM1/PGM3)

“…2‐Methyl‐(1,2‐dideoxy‐α‐ d ‐glucopyranoso)‐[2,1‐ d ]‐2‐oxazoline (13): , Compound 12 (1.05 g, 3.03 mmol) was dissolved in dry MeOH (0.1 m; 30 mL) under an inert atmosphere. NaOMe (6 equiv.)…”

Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T

“…There are a few reports on LNT2 formation by GH20 catalyzed transglycosylation with (GlcNAc) 2 or pNPGlcNAc as donors and lactose as the acceptor [15,18,64] (see Figure 6). Hydrolysis of LNT2 by PhNah20A, an HMO core structure [70], warranted the investigation of the transglycosylation with (GlcNAc) 2 and the GH20 reaction intermediate NAG-oxazoline [2-methyl-(1,2-dideoxyα-d-glucopyrano)-oxazoline] [71] as a donor and lactose as an acceptor ( Figure 7A). We here also demonstrated transglycosylation by the commercial GH20 N-acetylglucosaminidase from S. plicatus (SpHex) [38] (see Figure S6), which has not been previously reported.…”

“…NAG-oxazoline [2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline] was synthesized and purified as described previously [71]. Briefly, 2 g GlcNAc (9 mmol) was dissolved in 20 mL acetic anhydride, then we added 10 mL pyridine and stirred overnight at room temperature (RT).…”

“…Reaction mixtures for transglycosylation contained either 100 mM NAG-oxazoline (from 1 M stock in 50 mM sodium borate, pH 9.3) or 100 mM (GlcNAc) 2 donor, and as acceptor 200 mM lactose, d-galactose, d-glucose, 2-deoxy-d-glucose or l-fucose; 1 or 10 U•mL −1 (11.6 or 116 µg•mL −1 ) PhNah20A or 10 U•mL −1 S. plicatus β-NAHA in 50 mM sodium phosphate, pH 8.0, 0.5% BSA, at 37 • C. The reaction volume was typically 20 µL for TLC analysis and 250 µL for product yield and structure determination. Slightly basic conditions were required as NAG-oxazoline is not stable at neutral or acidic pH [71]. Reactions were stopped at various time points by heating (5 min, 90 • C), cooled to RT and centrifuged (12,000× g, 1 min, 4 • C).…”

Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal how lipase function depends on membrane curvature

“…To support this plethora of functionalities lipases have been extensively engineered 12,13 , to meet the demand for increasingly specific chemical reactions 14 . Our capacity to optimize or alter protein function has radically improved through site directed mutagenesis of the active site 15,16 , directed evolution 17 , generation of novel functions using molecular dynamics (MD) simulations 18 or machine learning 17 . The advancement of high throughput methodologies on the other hand may provide large numbers of mutants that have to be screened for the desired properties.…”