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Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations.

ABSTRACT: The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here, we report how the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally guided protein and substrate engineering, and applied to the oxidation of unactivated methylene C-H bonds. Molecular dynamics and quantum mechanical calculations were used to develop a predictive model for substrate scope, site selectivity and stereoselectivity of PikC-mediated C-H oxidation. A suite of menthol derivatives was screened computationally and evaluated through in vitro reactions, where each substrate adhered to the predicted models for selectivity and conversion to product. This platform was also expanded beyond menthol-based substrates to the selective hydroxylation of a variety of substrate cores ranging from cyclic to fused bicyclic and bridged bicyclic compounds.

SUBMITTER: Narayan AR

PROVIDER: S-EPMC4518477 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations.

Narayan Alison R H AR Jiménez-Osés Gonzalo G Liu Peng P Negretti Solymar S Zhao Wanxiang W Gilbert Michael M MM Ramabhadran Raghunath O RO Yang Yun-Fang YF Furan Lawrence R LR Li Zhe Z Podust Larissa M LM Montgomery John J Houk K N KN Sherman David H DH

Nature chemistry 20150629 8

The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here, we report how the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally guided protein and substrate engineering, and applied to the oxidation of unactivated methyle ...[more]

PMID: 26201742

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Project description:BACKGROUND:Free energy simulations are an important tool in the arsenal of computational biophysics, allowing the calculation of thermodynamic properties of binding or enzymatic reactions. This paper introduces methods to increase the accuracy and precision of free energy calculations by calculating the free energy costs of constraints during post-processing. The primary purpose of employing constraints for these free energy methods is to increase the phase space overlap between ensembles, which is required for accuracy and convergence. METHODS:The free energy costs of applying or removing constraints are calculated as additional explicit steps in the free energy cycle. The new techniques focus on hard degrees of freedom and use both gradients and Hessian estimation. Enthalpy, vibrational entropy, and Jacobian free energy terms are considered. RESULTS:We demonstrate the utility of this method with simple classical systems involving harmonic and anharmonic oscillators, four-atomic benchmark systems, an alchemical mutation of ethane to methanol, and free energy simulations between alanine and serine. The errors for the analytical test cases are all below 0.0007kcal/mol, and the accuracy of the free energy results of ethane to methanol is improved from 0.15 to 0.04kcal/mol. For the alanine to serine case, the phase space overlaps of the unconstrained simulations range between 0.15 and 0.9%. The introduction of constraints increases the overlap up to 2.05%. On average, the overlap increases by 94% relative to the unconstrained value and precision is doubled. CONCLUSIONS:The approach reduces errors arising from constraints by about an order of magnitude. Free energy simulations benefit from the use of constraints through enhanced convergence and higher precision. GENERAL SIGNIFICANCE:The primary utility of this approach is to calculate free energies for systems with disparate energy surfaces and bonded terms, especially in multi-scale molecular mechanics/quantum mechanics simulations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Dataset Information

Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations.

Publications

Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations.

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