Role of Active Site Rigidity in Activity: MD Simulation and Fluorescence Study on a Lipase Mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site

A Hierarchical Approach to Protein Fold Prediction

Fold recognition, assigning novel proteins to known structures, forms an important component of the overall protein structure discovery process. The available methods for protein fold recognition are limited by the low fold-coverage and/or low prediction accuracies. We describe here a new Support Vector Machine (SVM)-based method for protein fold prediction with high prediction accuracy and high fold-coverage. The new method of fold prediction with high fold-coverage was developed by training and testing on a large number of folds in order to make the method suitable for large scale fold predictions. However, presence of large number of folds in the training set made the classification task difficult as a consequence of increased complexity involved in binary classifications of SVMs. In order to overcome this complexity we adopted a hierarchical approach where fold-prediction is made in two steps. At the first step structural class of the query is predicted and at the second step fold is predicted within the predicted structural class. This decreased the complexity of the classification problem and also improved the overall fold prediction accuracy. To the best of our knowledge this is the first taxonomic fold recognition method to cover over 700 protein-folds and gives prediction accuracy of around 70% on a benchmark dataset. Since the new method gives rise to state of the art prediction performance and hence can be very useful for structural characterization of proteins discovered in various genomes

Active site dynamics of wild type and 6B lipase.

<p>(A) RMSD of C_α atoms of wild type and 6B lipases from their energy minimized crystal structures as a function of MD simulation time. For clarity, single simulation data is shown for both wild type and 6B lipase while others are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s003" target="_blank">Fig. S2A</a>. (B) RMSF of C_α atoms of individual residues during 2–20 ns simulation time. Active site residues positions are shown as solid spheres. (C) Typical time-resolved fluorescence anisotropic decay profiles of acrylodan attached to C77 in wild type and 6B lipase background.</p

Positions of active site residues and mutations on lipase molecule.

<p>Wild-type (grey) and 6B (pink) crystal structures were overlapped. Side chain of active site residues are shown as ball and stick in elemental color (labeled with black) on wild type scaffold, while side chains of mutations are shown as stick in pink color (labeled with pink) on 6B scaffold. Active site residues: I12, A15, F17, N18, S77, M78, A105, L108, D133, M134, I135, L140, G155, H156, I157, L160 and Y161 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188-van1" target="_blank">[18]</a>. Mutations: A15S, F17S, A20E, N89Y, G111D, L114P, A132D, M134E, M137P, I157M, S163P and N166Y. Stereo figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s002" target="_blank">Fig. S1</a>.</p

Catalytic mechanism of ester hydrolysis by <i>B. subtilis</i> lipase.

<p>Catalytic mechanism of ester hydrolysis by <i>B. subtilis</i> lipase.</p

Catalytic parameters of lipases at room temperature (∼20°C).

<p>Catalytic parameters of lipases at room temperature (∼20°C).</p

Active site geometry of wild type and 6B lipase during 2–20 ns MD simulations.

<p>(A) Frequency distribution of MD simulation structural snapshots as a function of distances between hydroxyl oxygen of S77 and imidazole nitrogen of H156. (B) Frequency distribution of MD simulation structural snapshots as a function of distances between imidazole nitrogen of H156 and carboxylate oxygen of D133. (C) Frequency distribution of MD simulation structural snapshots as a function of RMSD of their catalytically important atoms (hydroxyl oxygen of S77, imidazole nitrogens of H156, carboxylate oxygen of D133 and peptidic nitrogens of I12 and M78) to that of transition state analog bound crystal structure (PDB id: 1R4Z, Chain A).</p

Active site in transition state bound and free form.

<p>Structural overlap of active site of the free wild type lipase and in complex with covalently attached transition state analog (chain A of PDB id: 1I6W and 1R4Z). Transition state analog is O-[(R)-1,2-O-isopropylidene-sn-glycerol]6-hexenyl phosphonate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188-Labeikovsky1" target="_blank">[34]</a>. Free enzyme is shown in green while complex is shown in elemental color. Side chains are shown as sticks while backbone as lines. Stereo figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s007" target="_blank">Fig. S6</a>.</p