Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

Abstract

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates

“…Yields could be increased by varying the ratios of donor and acceptor, reaction times, and Hv LD concentrations. Another approach to increase yields could be through the mutation of selected residues in the active site, either the nucleophile to obtain a glycosynthase [ 31 ], or an evolutionary approach involving the mutation of conserved residues but excluding catalytic residues [ 32 , 33 ].…”

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima

“…The mgtA gene from T. maritima (GenBank accession number AAD35451.1), previously cloned in the plasmid pET22b(+) [ 52 ], was used as a template to build the two mutants of Tm GTase. The mutant genes were constructed by PCR using the corresponding pair of oligonucleotides for each mutation shown in Table 1 .…”