Abstract:The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding, and even dramatically impact enzyme function. When these flexible regions are structurally unresolvable, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here, we present a collaborative approach co… Show more
“…These results uncovered that different types of substituted groups at C4 of the indole ring can affect key parameters (activity and regio-selectivity) of TxtE biocatalysts. Interestingly, a single residue His176 in the F/G loop of TxtE was computationally identified as a potential determinant of enzyme regio-selectivity, and engineering of this site to some residues (e.g., Phe and Tyr) indeed created TxtE mutants that predominantly produce 5-NO 2 - l -Trp 44 . Our results provided new insights into intriguing and synthetically important TxtE’s regio-selectivity.Figure 4Both 4-Me-5-NO 2 - l -Trp and 4-Me-7-NO 2 - l -Trp were produced in the TB14 reaction with 4-Me- dl -Trp as substrate.…”
Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-dl-Trp. The production of both 4-Me-5-NO2-l-Trp and 4-Me-7-NO2-l-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.
“…These results uncovered that different types of substituted groups at C4 of the indole ring can affect key parameters (activity and regio-selectivity) of TxtE biocatalysts. Interestingly, a single residue His176 in the F/G loop of TxtE was computationally identified as a potential determinant of enzyme regio-selectivity, and engineering of this site to some residues (e.g., Phe and Tyr) indeed created TxtE mutants that predominantly produce 5-NO 2 - l -Trp 44 . Our results provided new insights into intriguing and synthetically important TxtE’s regio-selectivity.Figure 4Both 4-Me-5-NO 2 - l -Trp and 4-Me-7-NO 2 - l -Trp were produced in the TB14 reaction with 4-Me- dl -Trp as substrate.…”
Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-dl-Trp. The production of both 4-Me-5-NO2-l-Trp and 4-Me-7-NO2-l-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.
“…Arnold and coworkers obtained crystal structures of P450 TxtE that revealed a functional role of the B/C loop; however, they were unable to determine the interactions made by the F/G loop, as it is disordered in the crystal structure 149 . Computational approaches were used to model the structure and dynamics of the disordered F/G loop 150 . First, the F/G loop was constructed via homology modeling followed by short MD simulations.…”
Section: Engineering Functional Regions Into Proteinsmentioning
A grand challenge in the field of structural biology is to design and engineer proteins that exhibit targeted functions. Although much success on this front has been achieved, design success rates remain low, an ever-present reminder of our limited understanding of the relationship between amino acid sequences and the structures they adopt. In addition to experimental techniques and rational design strategies, computational methods have been employed to aid in the design and engineering of proteins. Molecular dynamics (MD) is one such method that simulates the motions of proteins according to classical dynamics. Here, we review how insights into protein dynamics derived from MD simulations have influenced the design of proteins. One of the greatest strengths of MD is its capacity to reveal information beyond what is available in the static structures deposited in the Protein Data Bank. In this regard simulations can be used to directly guide protein design by providing atomistic details of the dynamic molecular interactions contributing to protein stability and function. MD simulations can also be used as a virtual screening tool to rank, select, identify, and assess potential designs. MD is uniquely poised to inform protein design efforts where the application requires realistic models of protein dynamics and atomic level descriptions of the relationship between dynamics and function. Here, we review cases where MD simulations was used to modulate protein stability and protein function by providing information regarding the conformation(s), conformational transitions, interactions, and dynamics that govern stability and function. In addition, we discuss cases where conformations from protein folding/unfolding simulations have been exploited for protein design, yielding novel outcomes that could not be obtained from static structures.
“…An important feature of MD is that it is able to simulate the flexible active site of CYPs in the order of nanoseconds time scale, thus accounting for the flexibility of this tool. The technique allows exploration of thermal motion, binding conformations and binding affinities of the ligands, as well as the motion, induced fit and conformational changes of CYP substrate access channel and active pocket [31][32][33]. With MD combined, docking result can be further strengthened.…”
-In vitro and in silico models of drug metabolism are utilized regularly in the drug research and development as tools for assessing pharmacokinetic variability and drug-drug interaction risk. The use of in vitro and in silico predictive approaches offers advantages including guiding rational design of clinical drug-drug interaction studies, minimization of human risk in the clinical trials, as well as cost and time savings due to lesser attrition during compound development process. This article gives a review of some of the current in vitro and in silico methods used to characterize cytochrome P450(CYP)-mediated drug metabolism for estimating pharmacokinetic variability and the magnitude of drug-drug interactions. Examples demonstrating the predictive applicability of specific in vitro and in silico approaches are described. Commonly encountered confounding factors and sources of bias and error in these approaches are presented. With the advent of technological advancement in high throughput screening and computer power, the in vitro and in silico methods are becoming more efficient and reliable and will continue to contribute to the process of drug discovery, development and ultimately safer and more effective pharmacotherapy.
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