Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases. Issue 9 (7th June 2016)

• Record Type: Journal Article
• Authors: Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.; Broadbelt, Linda J.; Ståhlberg, Jerry; Beckham, Gregg T.
• Is Part Of: Chemical science. Volume 7:Issue 9(2016:Sep.)
• Page Start: 5955
• Page End: 5968
• Publication Date: 2016-06-07
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c6sc00571c
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : Unbiased simulations reveal a water wire enabling and rescuing the catalytic base of an inverting glycoside hydrolase. Abstract : In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei ( Hypocrea jecorina ) GH6 cellulase ( Tr Cel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleaving the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the −1 sugar adopts a 2 SO ring configuration as it reaches its binding site. This work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.