Y, Baghdad, Iraq, for any doctoral fellowship. Informed Consent Statement: Not applicable. Information Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.Molecules 2021, 26,23 of
moleculesArticleModulating Glycoside Hydrolase Activity involving Hydrolysis and Transfer Reactions Using an Evolutionary ApproachRodrigo A. Arreola-Barroso, Alexey Llopiz , Leticia Olvera and Gloria Ro 67-4853 site Saab-Rinc Departamento de Ingenier Celular y Biocat isis, Instituto de Biotecnolog , Universidad Nacional Aut oma de M ico, Cuernavaca 62271, Mexico; [email protected] (R.A.A.-B.); [email protected] (A.L.); [email protected] (L.O.) Correspondence: [email protected]: Arreola-Barroso, R.A.; Llopiz, A.; Olvera, L.; Saab-Rinc , G. Modulating Glycoside Hydrolase Activity amongst Hydrolysis and Transfer Reactions Employing an Evolutionary Strategy. Molecules 2021, 26, 6586. https://doi.org/ 10.3390/molecules26216586 Academic Editor: Stefan Janecek Received: 23 September 2021 Accepted: 28 October 2021 Published: 30 OctoberAbstract: The proteins inside the CAZy glycoside hydrolase loved ones GH13 catalyze the hydrolysis of polysaccharides which include glycogen and starch. Lots of of those enzymes also execute transglycosylation in numerous degrees, ranging from secondary to predominant reactions. Identifying structural determinants linked with GH13 family members reaction specificity is key to modifying and designing enzymes with increased specificity towards person reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational strategy 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 -amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima have been constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio although the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations in the variants indicated adjustments in flexibility which can account for the modified T/H ratio. An vital contribution of your presented computational strategy is its capacity to recognize residues outside of the active center that influence the reaction specificity. Keyword phrases: transglycosidation; hydrolysis; contact-residues; amylase; glucanotransferase; coevolution; enrichment-factor; specificity1. Introduction Enzymes are accelerators of chemical reactions that occur in living cells, which also work in vitro, producing their use in the laboratory, in healthcare applications, and in market attainable [1]. Tailoring an enzyme’s capacity to carry out precise reactions is one of the greatest challenges that has to be met in an effort to move on to a additional sustainable biocatalysis course of action [4]. Within this sense, directed evolution has confirmed to become a BMS-820132 Protocol valuable method for evolving functions, together with the limitation of requiring substantial screening efforts, as a way to find an improved biocatalyst [5,6]. De novo design has shown impressive improvements over the last two decades in the improvement of energy functions for directing the style of proteins [7]. Having said that, the subtle alterations that confer the essential dynamics for catalysis have not yet been determined.