A single point mutation alters the transglycosylation/hydrolysis partition, significantly enhancing the synthetic capability of an endo-glycoceramidase

Julien Durand (1) , Xevi Biarnés (2) , Laurie Watterlot (1) , Cyrielle Bonzom (1) , Vinciane Borsenberger (1) , Antoni Planas (2) , Sophie Bozonnet (1) , Michael O'Donohue (1) , Régis Fauré (1)

1 LISBP - Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés
2 Laboratory of Biochemistry, Institut Químic de Sarrià

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 beta-cellobioside, have provided appropriate conditions for the efficient synthesis of alkyl beta-cellobioside derivatives. The mutant D311Y was characterized by a lowered KM value for the hydrolysis of 2-chloro-4-nitrophenyl beta-cellobioside and increased transglycosylation when using aliphatic 1,3-diols or alcohols bearing a delta-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.

Mots clés: glycoside hydrolase; mutagenesis; transglycosylation; alkyl cellobiosides; molecular interactions; chemoenzymatic glycosynthesis

Domaines: Sciences du Vivant [q-bio]

Julien Durand, Xevi Biarnés, Laurie Watterlot, Cyrielle Bonzom, Vinciane Borsenberger, et al.. A single point mutation alters the transglycosylation/hydrolysis partition, significantly enhancing the synthetic capability of an endo-glycoceramidase. ACS Catalysis, 2016, 6 (12), pp.8264-8275. ⟨10.1021/acscatal.6b02159⟩. ⟨hal-01606355⟩