Alladi Ramakrishnan Hall

Understanding the critical role of electrostatic and non-electrostatic interactions in important biological systems.

Anagh Mukherjee

Saha Institute of Nuclear Physics

The investigation of the role of the electrostatics in systems of widespread interest employing
computational techniques is an emerging area of research. The influence of built-in local electric fields
(LEFs) present in naturally occurring macrocyclic systems such as valinomycin has been explored.
Static and ab initio molecular dynamics (AIMD) calculations indicate that LEFs are the primary driving
factor in determining the energetically favored position of counter anions such as chloride (Cl−) in the
potassium (K+) and sodium (Na+) coordinated valinomycin macrocycle structures: they exist inside the
cage in the case of K+ sequestration by valinomycin and outside for Na+. This divergence has been
proposed to be the determining factor for the selectivity of the valinomycin macrocycle for binding a
K+ cation over Na+.
The role of non-electrostatic interactions on the catalytic function of enzymes has not been studied
extensively. The artificial metalloenzyme containing an Iridium in place of
Iron along with four directed evolution C317G, T213G, L69V, V254L mutations in a natural
Cytochrome P450 presents an important milestone in merging the extraordinary efficiency of
biocatalyst with the versatility of small molecule chemical catalyst in catalyzing a new-to-nature
carbene insertion reaction. This is a show-stopper enzyme as it exhibits a catalytic efficiency similar to
that of natural enzymes. Despite this remarkable discovery, there is no mechanistic understanding as to
why it displays extraordinary efficiency after incorporation of the four active site mutations by directed
evolution methods, so far been intractable to any experimental methods. In this study, we have
deciphered the catalytically active conformation of this natural-like artificial metalloenzyme using
large-scale molecular dynamics simulations and rigorous quantum chemical calculations.Our study
reveals how directed evolution mutations precisely position the cofactor-substrate in an unusual but
effective orientation within a reshaped active site that emerged during evolution and stabilized by C‒
H...π interactions from more ordered mutated L69V and V254L residues. The active conformation
correctly reproduces the experimental barrier height and accounts for the catalytic effect of 2.7 kcal/mol,
in excellent agreement with experimental observations. Moreover, the active conformation features an
unusual bonding interaction in a metal-carbene species that preferentially stabilizes the rate determining
formation of an Iridium Porphyrin Carbene intermediate to render the observed high catalytic rate
acceleration. Despite the lack of any pre-existing structural data, our study maps the gradual evolution
of the substrate-cofactor conformational space with successive active site mutations to generate the
catalytically efficient conformation. While the electrostatic model is widely established for enzyme
catalysis, this study reports for the first-time an unprecedented catalytic role of non-electrostatic
interactions into it and suggests a new principle towards designing enzymes with natural efficiency.