(41c) Understanding the Role of Ionic Liquids in the Enzyme Catalyzed Breakdown of Cellulose Using Molecular Dynamics Simulations

Because of their
potential for single-pot cellulose dissolution and enzymatic degradation, Ionic
liquids (ILs) afford an opportunity to optimize the biocatalytic conversion of
non-food plant biomass into biofuels. Established methods for the pretreatment
of biomass are costly, inefficient, and often use harsh processing conditions
and chemical solvents resolving in unwanted by-products.¹ ILs possess unique properties (e.g. negligible flammability, and vapor
pressure), demonstrate wide chemical and thermal stability, and can be easily
recycled.² However, they have also been shown to
negatively affect the activity of many cellulases. With the goal of rapid
conversion of biomass to glucose, molecular level insight can assist in the
rational design of enzymes and ILs.

Progress has
been made to further understand IL-induced unfolding and deactivation^3,4,
but still little is known about how ILs influence the catalytic mechanisms of
these cellulases. Using molecular dynamics, these mechanisms can be probed at
an atomic level. The key challenge in modeling these systems is to find the
balance needed between computational cost and accuracy. The hybrid quantum
mechanics/molecular mechanics (QM/MM) approach is a method that can be used to
probe these enzymatic reactions with the strength of QM accuracy, and the speed
of MM forcefields. Still, the timescales needed to observe these phenomena
occur on much larger time scales than are computationally affordable. Enhanced
sampling techniques can be used to circumvent these timescale issues. For
example in metadynamics⁵, a powerful enhanced sampling algorithm, a
time dependent bias is applied to a few coarse slow degrees of freedom to enhance phase space exploration by
discouraging the system from visiting previous states.

This talk presents
our research using QM/MM molecular dynamics to model the biocatalytic breakdown
of cellulose. Using the enhanced sampling method well-tempered metadynamics⁶ we study the effect of ILs on the
thermodynamics of enzyme catalyzed reactions and determine the role ILs play on
the catalytic mechanism. These simulations feature an exemplary Cel5A retaining
endoglucanase, in complex with a cellotetraose ligand modeled in an aqueous IL
1-butyl-3- methylimidazolium chloride [BMIM][Cl] solution, Figure 1. The results
indicate the presence of an ILs in the active site in both the holo and apo
forms of the enzyme. The free-energy landscape for glycosylation suggests IL
inhibition of the intermediates formed in this two-step reaction. Broadly, this
provides us with a basis for understanding IL inhibition of Cel5A activity.

Figure 1. Showing the
presence of [BMIM][Cl] (colored by atom name in licorice), in the active site (orange,surf) of
Cel5A in complex with a cellulose ligand (green, licorice).

References

[1] P. Engel, R. Mladenov, H. Wulfhorst, G. Jager, & A. C. Spiess.
“Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of
native and modified cellulose”, Green
Chem., 2013, 12, 1959-1966.

[2] J. Serra Moreno, S. Jeremias, A. Moretti, S. Panero, S. Passerini, B. Scrosati, G.B. Appetecchi. “Ionic
liquid mixtures with tunable physiochemical
properties”, Electrochimica Acta., 2015, 599-608.

[3] S.R. Summers, K. G. Sprenger, J. Pfaendtner, J. Marchant, M.
F. Summers, J. L. Kaar, “Mechanism of Competitive
Inhibition and Destabilization of Acidothermus cellulolyticus Endoglucanase 1
by Ionic Liquids”, J. Phys. Chem. B.,
2017, 121, 10793−10803.

[4] V. Jaeger, P. Burney, J. Pfaendtner, “Comparison of Three
Ionic Liquid-Tolerant Cellulases by Molecular Dynamics”, Biophys. J., 2015, 108(4)
880–892.

[5] A. Laio and M. Parrinello. “Escaping free energy
minima”, Proc. Natl. Acad. Sci. USA, 2002, 99:12562–12566.

[6] A. Barducci, G. Bussi, M. Parinell, “Well-Tempered Metadynamics: A Smoothly
Converging and Tunable Free-Energy Method”, Phys.
Rev. Lett, 2008, 100(2), 020603.