Engineering three GH84 enzymes into glycoside phosphorylases and understanding the nature of the reaction intermediate

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Engineering three GH84 enzymes into glycoside phosphorylases and understanding the nature of the reaction intermediate

David Teze 1,2 , Joan Coines 3 , Lluis Raich 3 , Valentina Kalichuk 2 , Claude Solleux 2 , Charles Tellier 2 , Corinne Miral 2 , Carme Rovira 3 , Birte Svensson 1

1 Technical University Of Denmark, Kgs. Lyngby, Denmark, 2 University of Nantes, Nantes, France, 3 University of Barcelona, Barcelona, Spain

Carbohydrate active enzymes (CAZymes) include glycoside hydrolases (GH), auxiliary activities, e.g. oxidases (AA), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE) and glycoside phosphorylases. Contrarily to the other enzyme categories, glycoside phosphorylases do not have their own separate classification but are mingling within GT (GT4, 35) and GH (GH3, 13, 65, 94, 112, 130) [1]. However, the conversion of a GH or a GT into a glycoside phosphorylase has yet to be reported. Here, we converted three bacterial GH84s from Streptococcus pyogenes (SpOGA), Oceanicola granulosus (OgOGA) and Thermobaculum terrenum (TtOGA) (sharing 29−33% identity pairwise) into glycoside phosphorylases. While OgOGA and TtOGA variants display both hydrolysis and phosphorolysis, SpOGA became a pure phosphorylase with a reasonable activity having kcat = 2.7 ± 0.2 s–1, which is comparable with the only known natural retaining β-glycoside phosphorylases, that belongs to GH3 [2].

To understand what enabled this conversion, metadynamics QM(DFT)/MM simulations were performed on TtOGA and its variant. Our calculations reproduced the experimental data and showed that the electrostatic potential in the active site is the main driver towards the specificity shift. The computed molecular mechanism of hydrolysis/phosphorylation showed that both mutant and native enzymes feature an oxazolinium ion -and not a neutral oxazoline [3,4]- as an intermediate. Understanding the molecular details of the GH84 mechanism is important for the design of inhibitors against the clinically relevant human GH84. Indeed, GH84 are involved in protein regulation through O-GlcNAc removal on Ser/Thr, a modification that controls protein (in)activation and phosphorylation [5].

Grants from MINECO and the Novo Nordisk foundation supported this research.

Kitaoka, M. Diversity of phosphorylases in glycoside hydrolase families. Appl. Microbiol. Biotechnol. 2015, 99, 8377–8390.

Macdonald, S. S., Blaukopf, M. & Withers, S. G. N-acetylglucosaminidases from CAZy family GH3 are really glycoside phosphorylases, thereby explaining their use of histidine as an acid/base catalyst in place of glutamic acid. J. Biol. Chem. 2015, 290, 4887–4895.

Coines, J., Alfonso-Prieto, M., Biarnés, X., Planas, A. & Rovira, C. Oxazoline or oxazolinium ion? the protonation state and conformation of the reaction intermediate of chitinase enzymes revisited. Chem. Eur. J. 2018, 24, 19258–19265.

Vocadlo, D. J. O-GlcNAc processing enzymes : catalytic mechanisms , substrate specificity , and enzyme regulation. Curr. Opin. Chem. Biol. 2012, 16, 488–497.

Hart, G. W., Housley, M. P. & Slawson, C. Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 2007, 446, 1017–1022.

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