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 beta-xylobioside with the retaining endo-beta-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 (4)C(1) chair conformation shows spontaneous transformation to the (2,5)B boat conformation, and potential of mean force calculations indicate that the boat is approximately 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 (4)C(1) 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 beta-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the (4)C(1) 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 (2,5)B conformation.

Direct Experimental Evidence for the High Chemical Reactivity of α‐ and β‐Xylopyranosides Adopting a 2,5B Conformation in Glycosyl Transfer

“…[14] Such a conformation is attained more easily for xylosides than for glucosides owing to the lack of a bulky hydroxymethyl substituent, which is forced into a pseudo-A C H T U N G T R E N N U N G axial orientation in the latter case. This could explain the high specificity of the GH-11 xylanase family for xylosyl units, in contrast to the more promiscuous behavior of the GH-10 xylanase family, which utilizes a 4 C 1 conformation for the intermediate.…”

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

“…5,6 The conformational interconversions that occur in the active sites of specific GHs have been the subject of many recent studies. [7][8][9][10][11][12][13][14] Nevertheless, direct evidence of conformational itineraries is still missing for many GH families. Furthermore, detailed structural features of the enzymatic transition state (TS) in GHs are still fairly unknown.…”

Catalysis: transition-state molecular recognition?

“…5): the conformational change is accompanied by a marked decrease in the length of the O Y H Y … O ring hydrogen bond. Moreover, analogous simulations for the Tyr69Phe mutant (lacking O Y ) showed the chair to be stable, thereby confirming the key role of Tyr69 in preferentially stabilising the boat, with a relative free energy difference of about 20 kJ mol −1 , by means of the O Y H Y … O ring hydrogen bond [23]. …”

“…MD simulations with the hybrid AM1/OPLS-AA/TIP3P method showed that both 4 C 1 chair and 2,5 B boat conformers of phenyl β-xyloside remained stable in water during the course of 30 ps trajectories, even in the presence of propionate and propionic acid moieties to mimic Glu78 and Glu172 [23]. In contrast, analogous MD simulations for the 4 C 1 conformer of the reactant complex of phenyl β-xylobioside with BCX showed spontaneous transformation to the 2,5 B conformer (Fig.…”