Development of molecular tools to provide mechanistic insights into diverse glycosidases

© 2016 Dr. Gaetano SpecialeGlycoside hydrolases are enzymes that catalyze the scission of the glycosidic bond. Despite the simplicity of the reaction they exist in a remarkable variety, numbering more than two hundred thousand known gene sequences grouped into more than 130 sequence related families. Understanding the unique features of each enzyme family is a key strategy for their utilization in biotechnology and for rational approaches to the treatment of diseases that involve glycoside hydrolases. The first part of this thesis involves the study of enzymes of glycoside hydrolase family 99 (GH99), a family of endo-α-1,2-mannosidases/endo-α-1,2-mannanases that have been proposed to undertake catalysis through a 1,2-anhydro sugar intermediate. A set of isotopically-labelled mannosides is synthesized and used for the measurements of kinetic isotope effects (KIEs) for basic solvolysis of 4-nitrophenyl α-D-mannopyranoside, which is known to proceed through a 1,2-anhydro sugar. The measured kinetic isotope effect (KIE) for H1, H2, C1, C2, O1 and O2 are 1.112, 1.045, 1.026, 0.999, 1.040, and 1.044, respectively. The transition state for the hydrolysis reaction was modeled with ab initio theoretical methods using the experimental KIE values as constraints. The results are consistent with the reaction mechanism involving rate-limiting formation of a transient 1,2-anhydro sugar that opens in water to give α-D-mannopyranose. These studies provide insight into the nature of the transition state of a bonafide neighboring group participation involving a 1,2-anhydro sugar and benchmark data for interpreting equivalent KIE data for the family GH99 mechanism. Next, a series of unlabelled and five-isotopically-labelled tetrasaccharides are synthesized and used to gain insight in the mechanism of a bacterial GH99 with a combination of protein crystallography, computational modelling and KIE measurements. The X-ray complex between a tetrasaccharide and a catalytically-inactive acid/base mutant enzyme shows an undistorted 4C1 conformation in the –1 subsite of the ground-state Michaelis complex. Using this complex as an input, computational modelling of the enzymic reaction leading to product revealed a 4C1 ↔ [4E]‡ ↔ 4H5 conformational itinerary. The measured H1-KIE value for α-2H was 1.134 (± 0.009), suggesting a dissociative, early TS. These data are collectively consistent with the proposed neighboring group participation mechanism. Compelling evidence for or against this mechanism, with an interpretation supported by the benchmark data for the chemical solvolysis process, should be available when KIE measurements for the enzyme-catalyzed reaction are complete. The second part of the thesis involves the study of the poorly characterized endo-α-1,6-mannosidases of family GH76, and their involvement in the degradation of dietary fungal α-mannan. Using substrates and inhibitors synthesized for the purpose, a comprehensive study of the mechanism of these enzymes is undertaken, supported by site-directed mutagenesis, X-ray crystallography and computational free energy landscape analysis. This work reveals that enzymes of this family operate through a classical Koshland retaining mechanism and follow a typical mannosidase conformational itinerary of OS2 ↔ [B2,5]‡ ↔ 1S5, with the peculiarity of an hydrogen bond of the substrate 3-OH to the incoming/departing nucleophilic carboxylate. The final part of the thesis reports the discovery and characterization of the first dedicated sulfoquinovosidase, YihQ from E. coli, a member of glycoside hydrolase family GH31. Sulfoquinovosidase activity is necessary for the liberation of the sulfonated sugar sulfoquinovose from the sulfolipid sulfoquinovoside diacylglycerol, thus releasing sulfoquinovose for catabolism through the sulfoglycolysis pathway. Synthesis of the chromogenic substrate 4-nitrophenyl α-D-sulfoquinovoside and its use in kinetic assays allowed demonstration that YihQ is retaining glycosidase and the first dedicated α-sulfoquinovosidase. Kinetic and structural analysis of a series of enzyme mutants led to identification the catalytic residues and a triad of amino acids important for sulfonate binding and catalysis. Sequence analysis revealed YihQ orthologs to be widespread in bacteria, fungi, protozoa, metazoan and plants and thus an important gateway enzyme to the catabolic arc of the biogeochemical sulfur cycle