Computational mutagenesis reveals the role of active-site tyrosine in stabilising a boat conformation for the substrate: QM/MM molecular dynamics studies of wild-type and mutant xylanases

Abstract

Molecular dynamics simulations have been performed for non-covalent complexes of phenyl β-xylobioside with the retaining endo-β-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutant using a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme–substrate complex with the proximal xylose ring bound at the –1 subsite (adjacent to the scissile glycosidic bond) in the ⁴C₁ chair conformation shows spontaneous transformation to the ^2,5B boat conformation, and potential of mean force calculations indicate that the boat is ∼30 kJ mol⁻¹ lower in free energy than the chair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 in stabilizing the boat in preference to the ⁴C₁ chair conformation, with a relative free energy difference of about 20 kJ mol⁻¹, by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring. QM/MM MD simulations for phenyl β-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the ⁴C₁ chair to be stable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moiety seems to provide some stabilization for the ^2,5B conformation.