Step 1 of our approach is to determine “near attack” conformations of the substrate for the reaction by initial interaction studies, and conduct Molecular Dynamics (MD) simulation of the E-S complexes obtained.
Step 2 entails post simulation analyses that are carried out to explore the distribution of the substrate conformation in the enzyme’s entrance tunnel and the active site.
In step 3, the MD trajectories are analyzed to determine the atomic contacts between the enzyme and the substrate to obtain a consolidated list of residues for the Computational alanine scanning (CAS). In cases of bulky substrates, the conformations of the substrates may form clusters, which are separated by barriers hindering the substrate to move towards the active site. Representative E-S conformations from MD simulations from the tunnel entrance to active site are subjected to semi-empirical QM calculations to derive the geometries and energy profiles of the conformational transitions at the barriers. Computational alanine scanning using the snapshots derived from these calculations identifies the hotspot residues affecting the barrier transitions.
In step 4, these are then subjected to in-silico mutagenesis.
In step 5, the semi-empirical QM-based computational alanine scanning (QM-CAS) was carried out by incorporating amino acid substitutions in the extracted coordinates of the high energy barrier conformations. The enzyme variants are screened and ranked using QZyme DesignerTM. The variants are ranked according to the E-S interaction energies for the transition into the active site to its near attack conformation, and substrate distances to the catalytic site.
The main objective of “QZyme WorkBenchTM ” is to reduce time taken for execution of Enzyme Engineering Projects from 4+ months to 3 months or less and enable scientists to handle multiple projects at a given time. Some of the other objectives include
Abstracting IT related tasks from Scientists,
Managing efficient Server utilization.
Delight the Customer with Prompt Deliverables.
The enzyme penicillin G acylase (PGA; EC 22.214.171.124) is a heterodimeric protein consisting of a small subunit and a large subunit, which are formed by the processing of a single polypeptide precursor.
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