Computational Protein Engineering Research Articles

Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

AbstractThe R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.

Developing improved MD codes for understanding processive cellulases

The mechanism of action of cellulose-degrading enzymes is illuminated through a multidisciplinary collaboration that uses molecular dynamics (MD) simulations and expands the capabilities of MD codes to allow simulations of enzymes and substrates on petascale computational facilities. There is a class of glycoside hydrolase enzymes called cellulases that are thought to decrystallize and processively depolymerize cellulose using biochemical processes that are largely not understood. Understanding the mechanisms involved and improving the efficiency of this hydrolysis process through computational models and protein engineering presents a compelling grand challenge. A detailed understanding of cellulose structure, dynamics and enzyme function at the molecular level is required to direct protein engineers to the right modifications or to understand if natural thermodynamic or kinetic limits are in play. Much can be learned about processivity by conducting carefully designed molecular dynamics (MD) simulations of the binding and catalytic domains of cellulases with various substrate configurations, solvation models and thermodynamic protocols. Most of these numerical experiments, however, will require significant modification of existing code and algorithms in order to efficiently use current (terascale) and future (petascale) hardware to the degree of parallelism necessary to simulate a system of the size proposed here. This work will develop MD codes that can efficiently use terascale and petascale systems, not just for simple classical MD simulations, but also for more advanced methods, including umbrella sampling with complex restraints and reaction coordinates, transition path sampling, steered molecular dynamics, and quantum mechanical/molecular mechanical simulations of systems the size of cellulose degrading enzymes acting on cellulose.

Predicting an Improvement of Secondary Catalytic Activity of Promiscuos Isochorismate Pyruvate Lyase by Computational Design

Improvement of the secondary catalytic activity of promiscuous enzymes can be guided by computational protein engineering. This methodology has been applied to isochorismate pyruvate lyase (IPL) that catalyzes isochorismate transformation into pyruvate and salicylate but it also presents secondary activity catalyzing the transformation of chorismate into prephenate. According to the computational results, a mutation of Val by Ile at position 38 would keep the enol pyruvyl moiety of the substrate in a diaxial conformation, closer to the TS geometry, thus reducing the free energy barrier of the chemical reaction 4.4 kcal·mol-1 with respect to the native IPL.

Study of Conformation of Actin Monomer by Molecular Mechanics Calculation.

In this paper, the 3-D molecular structure of actin monomer (G-actin) is studied by using the molecular mechanics analysis code, AMBER, which was developed by Kollman et al. . Actin and myosin molecules come together to construct the actomyosin system, which generates the motility assay to induce the whole muscle contraction. Several constitutive models for the muscle material exist under the theoretical basis of continuum mechanics, but the contraction process model is still incomplete. Therefore, we focus the investigation on the clarification of the contraction mechanism at the microlevel, because, in this decade there has been great progress in computational chemistry and protein engineering at the molecular level. The conformation of the actin molecule in vivo should take the optimum structure to generate the relative sliding between actin and myosin filaments under ATPase hydrolysis. At this moment the molecular structure of actin may be optimized in the molecular mechanics sense. We employ the molecular structure data of G-actin determined experimentally by Holmes et al. in 1990. The minimum-energy conformations of G-actin in four cases, e. g., with ATP or without ATP, and proK-actin-cut off between Met-47 and Gly-48- or normal actin, were determined by the steepest-descent method and the conjugate-gradient method in the molecular mechanics calculation. We investigate the differences of the conformation and potential energies among these four cases to characterize the atom structure and mechanical properties.