Sammanfattning

The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle.

Publicerad i

Journal of Biological Chemistry
2018, Volym: 293, nummer: 13, sidor: 4680-4687
Utgivare: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

SLU författare

• Ståhlberg, Jerry

  • Institutionen för molekylära vetenskaper, Sveriges lantbruksuniversitet
    

UKÄ forskningsämne

Biokemi och molekylärbiologi
Bioenergi


Publikationens identifierare

DOI: https://doi.org/10.1074/jbc.RA117.001186

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/94919