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Functional and Mechanistic Analyses of Biomimetic Aminoacyl Transfer Reactions in de Novo Designed Coiled Coil Peptides via Rational Active Site Engineering
journal contribution
posted on 2007-03-14, 00:00 authored by Luke J. Leman, Dana A. Weinberger, Zheng-Zheng Huang, Keith M. Wilcoxen, M. Reza GhadiriRibosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis
through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of
coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular
aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer
mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from
adjacent noncovalently associated helical modules. In our designs peptide self-assembly positions a cysteine
residue that is used to covalently capture substrates from solution via transthiolesterification (substrate
loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a
covalently tethered amino acid or modeled by the ε-amine of an active site lysine. However, through
systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that
the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to ∼103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and
variations in the inter-residue distances and geometry. Mechanistic studies based on 15N NMR and kinetic
analysis further indicate that certain active site constellations furnish an unexpectedly large pKa depression
(1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl
transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient
de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes.