title: Theoretical Insights into the Reaction and Inhibition Mechanism of Metal-Independent Retaining Glycosyltransferase Responsible for Mycothiol Biosynthesis.
abstract: Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus, the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center, and therefore, they are classified as either inverting or retaining GTs. While the inverting GT reaction mechanism has been widely studied, the retaining GT mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNANin IUPAC nomenclature) and has a 17.5 kcal/mol free energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion-like transition state. In contrast, fluorinated substrate analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the transition state.
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.|Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Juan I
authors.initials: JI
authors.lastname: Blanco Capurro
authors.affiliation: Department of Physics, University of Florida , Gainesville, Florida 32611, United States.
authors.forename: Chad W
authors.initials: CW
authors.lastname: Hopkins
authors.affiliation: Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes , Sáenz Peña 352, Bernal B1876BXD, Argentina.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Pierdominici Sottile
authors.affiliation: Departamento de Quimica Inorgánica, Anlı́tica y Quı́mica Fı́sica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.|Insituto de Quimica Inorgánica, Materiales Ambiente y Energı́a (INQUIMAE), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Mariano C
authors.initials: MC
authors.lastname: González Lebrero
authors.affiliation: Department of Chemistry, University of Florida , Gainesville, Florida 32611, United States.
authors.forename: Adrian E
authors.identifier: 0000-0003-3963-8784
authors.initials: AE
authors.lastname: Roitberg
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.|Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET, Ciudad Universitaria , Intendente Guiraldes 2160, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Marti
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D055162:Biocatalysis; D003545:Cysteine / Q000096:biosynthesis* / Q000737:chemistry; D006020:Glycopeptides / Q000096:biosynthesis* / Q000737:chemistry; D016695:Glycosyltransferases / Q000378:metabolism*; D007294:Inositol / Q000096:biosynthesis* / Q000737:chemistry; D008670:Metals / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D011789:Quantum Theory
chemical_list: D001426:Bacterial Proteins; D006020:Glycopeptides; D008670:Metals; C089265:mycothiol; D007294:Inositol; D016695:Glycosyltransferases; D000581:Amidohydrolases; C417275:N-acetyl-1-D-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase; D003545:Cysteine