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A single point mutation alters the hydrolysis/transglycosylation partition, significantly enhancing the synthetic capability of an endo-glycoceramidase.
J. Durand, X..Biarnés, L. Watterlot, C. Bonzom, V. Borsenberger, A. Planas, S. Bozonnet, M.J. O’Donohue, R. Fauré.
ACS Catalysis 6, 8264–8275 (2016). http://dx.doi.org/10.1021/acscatal.6b02159

Abstract

The mutation of D311 to tyrosine in endo-glycoceramidase II from Rhodococcus sp. and the use of a poorly recognized substrate, 2-chloro-4-nitrophenyl β-cellobioside, have provided appropriate conditions for the efficient synthesis of alkyl β-cellobioside derivatives. The mutant D311Y was characterized by a lowered KM value for the hydrolysis of 2-chloro-4-nitrophenyl β-cellobioside and increased transglycosylation when using aliphatic 1,3-diols or alcohols bearing a δ-hydroxy ketone function as acceptors. Closer analysis revealed that the transglycosylation/hydrolysis ratio in reactions catalyzed by the mutant was completely inversed and weak secondary hydrolysis was postponed, thus providing the basis for high transglycosylation yields (between 68 and 93%). Overall, results confirm that the enhancement of transglycosylation in glycoside hydrolases can be achieved by a combination of destabilized transition states and increased recognition for acceptor molecules.