NDLI: Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

Content Provider	Royal Society of Chemistry (RSC)
Author	Crowley, Michael F. Beckham, Gregg T. Knott, Brandon C. Mayes, Heather B. Ståhlberg, Jerry Broadbelt, Linda J.
Copyright Year	2016
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 (TrCel6A), 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 2SO 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.
Starting Page	5955
Ending Page	5968
Page Count	14
File Format	HTM / HTML PDF
ISSN	20416520
Volume Number	7
Issue Number	9
Journal	Chemical Science
DOI	10.1039/c6sc00571c
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Subscribed
Subject Keyword	Alanine Hydrolase Quantum chemistry Enzyme Active site Cellulose Hypocrea Trichoderma reesei Protein Molecular mechanics Hydrolysis Glycosidic bond Cellulase Transition path sampling Grotthuss mechanism Proton Carbohydrate Mutation Hydrogen bond
Content Type	Text
Resource Type	Article
Subject	Chemistry

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