George Osanjo 1-3*, Eva Aluvaala5, Meshack Wadegu5, Wallace Bulimo4,5, Anastasia N. Guantai1,3, Faith A. Okalebo1,3, Margaret Oluka1,3, Francis Mulaa2,4. Carbohydrate – active enzymes (Cazymes) as drug targets and tools for synthesis of medicinal compounds
Glycans and the enzymes that modify them – the carbohydrate active enzymes (Cazymes)- play key roles in biomolecular communication and in disease pathogenesis. Essential biological recognition information encoded by glycans includes the human ABO blood group determinants which mediate transfusion rejection reactions, promotion of neurogenesis, mediation of the inflammatory response and infection by viruses such as the Influenza A virus. Neuraminidases and alpha-L-fucosidases are Cazymes that catalyse the removal of terminal sialic acid and fucose residues from glycans respectively.
We explored the structure/function relationships of Cazymes and their glycan ligands using the neuraminidase and alpha-L-fucosidase (TmFuc) from Influenza A virus and Thermotoga maritima respectively.
The Influenza A H3N2 viral neuraminidase was obtained by generating cDNA using a reverse transcriptase, followed by polymerase chain reaction, gene cloning and expression in Escherichia coli XL1 blue cells. alpha-L-fucosidase (TmFuc) was cloned from the hyperthermophile Thermotoga maritima.
Directed molecular evolution approach was used to engineer alpha-L-fucosidase (TmFuc) to improve its transglycosidase activity relative to hydrolytic function. Up to now enhancement of transglycosylation activities of natural glycosidases has been obtained by directed mutagenesis or by protein engineering of various exoglycosidases or endoglycosidases.
Transferase activity of the Cazymeswere assessed by nuclear magnetic resonance (NMR) and capillary electrophoresis.
Wild-type TmFuc catalysed oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-2) regioselectivity at low yields (7 %). Similar transglycosidase activity was not observed with the Influenza A virus neuraminidase.
Following a cycle of mutagenesis and in vitro recombination of wild-type Tmfuc, it was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60 % of the overall wild type enzyme activity. Such synthetic improvements were obtained with only three mutations (T264A, Y267F, L322P) which were all located in the second amino-acid shell of the fucosidase active site.
Discussion and Conclusion
Molecular modeling suggested that some of these mutations (T264A, Y267F) of Tmfuc cause a reorientation of the amino-acids that are in direct contact with the substrates resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for synthesis of fuco-oligosaccharides. While the Influenza A virus neuraminidase did not show trans-sialidase activity, the methodology developed could be used to evaluate susceptibility of Influenza A virus to neuraminidase inhibitors, the current first line medicines for management of influenza A virus infections.
1st International Scientific Conference
Publication Author(s): DR. OSANJO GEORGE OYAMO