In silico characterisation and catalytic property analysis of L-2-Haloacid dehalogenase (DEHL) from rhizobium SP.RC1

Abstract
L-2-haloacid dehalogenases (DehL) (EC 3.8.1.2) are of high interest due to their potential application in bioremediation and in synthesis of various industrial products. Imperatively, high quantities of halogen-based organic compounds are synthesised biogenically as well as formed anthropogenically, with the latter being the bigger contributor. Limiting the manufacturing activities would not be an economically feasible option to reduce the accumulation of such recalcitrant compounds in the biosphere. One such way to overcome this issue is to rationally engineer the existing L-specific dehalogenases into D-specific ones, as L-specific native dehalogenases are unable to degrade the D-isomers of the compounds that are formed anthropogenically. For this present study, DehL of Rhizobium sp. RC1 was chosen as the model enzyme, as the natural habitat of the bacterium is soil. Since the molecular structure of DehL is not available, as well as its catalytic mechanism, the study employed a multi-template threading approach to generate the first computational model of DehL ever. Experimentally solved structures of other L-2- haloacid dehalogenases (PDB: 1JUD, 1ZRN, 3UM9, 1AQ6, 3UMB and 2NO4) were used as the templates. The structure of DehL consists of a-helices and ß-sheets organised into two domains namely: main domain and sub-domain. Then, using a combination of ab initio fragment molecular orbital (FMO) calculations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations and classical molecular dynamic simulation (MD) of the three-dimensional model of DehL-L-2- chloropropionic acid complex, the catalytic residues of DehL were predicted. The individual in silico replacements of Asp13, Thr17, Met48, Arg51 and His184 with alanine were found to significantly decrease the binding free energy for the DehL-L- 2-chloropropionate complex, indicating their involvement in the catalytic mechanism, as mutation to alanine delete the function of such residues. Strong interfragment interaction energies (IFIEs) calculated for Asp13 and L-2-chloropropionic acid, and for a water molecule (WT1) and His184 were estimated. Retention of distances between atoms in the aforementioned pairs during MD runs corresponded well with the role of Asp13 as the nucleophile and His184 being the essential molecule or basic residue to activate the catalytic water during DehL catalysis. Subsequent investigation using quantum mechanics/molecular mechanics (QM/MM) and binding energy calculations, revealed R51L mutation led to the loss of DehL dehalogenation activity towards its natural substrate, L-2-chloropropionate, whereas M48R mutation caused DehL to gain activity toward the unnatural substrate, D-2- chloropropionate. In a nutshell, this study theoretically established the molecular mechanism of DehL dehalogenation. The amino acid residues responsible for the inversion of stereospecificity of DehL were also identified. The results presented here can potentially serve as the fundamental knowledge for rational design of DehL with improved efficiency and substrate utility, which may play important roles in the industry and environmental remediation
Description
Thesis (PhD. (Biosciences))
Keywords
Bioremediation—Research, Catalysts—Analysis
Citation