Structural and functional studies of non-stereospecific α-haloacid dehalogenase (DehE) from rhizobium sp. RC1 /
Environmental pollution caused by the abundance of xenobiotic compounds in nature. For instance, haloalkanoic acid substances released by agriculture activities were accumulated in our ecosystem and proven harmful to the living organisms. Fortunately, haloacid dehalogenase enzymes could catalysed th...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Science, International Islamic University Malaysia,
2014
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| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
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| Summary: | Environmental pollution caused by the abundance of xenobiotic compounds in nature. For instance, haloalkanoic acid substances released by agriculture activities were accumulated in our ecosystem and proven harmful to the living organisms. Fortunately, haloacid dehalogenase enzymes could catalysed the removal of halides from haloalkanoic acids and convert them into a harmless form. This study presents the structure conformation, functional residues and substrate affinity of the non-stereospecific α-haloacid dehalogenase (DehE) from Rhizobium sp. RC1. The three-dimensional structure of DehE was modelled by homology modelling using DehI from Pseudomonas putida PP3 (PDB ID: 3BJX, 72% identity) as the crystal structure template. Using computational analyses, a total of 12 active site residues were identified in DehE; whereby, 10 of these residues (W34, F37, F268, N114, Y117, Y265, S188, D189, I269, and I272) were verified using site-directed mutagenesis. DehE and its mutants were generated on expression vector pET-22b(+) using Escherichia coli host BL21. All of the 10 residues were affected by mutation showing diminishing specific activities of DehE down to below 80%. Meanwhile, a total loss of activity (0 %) at residue D189 had proven a direct attack mechanism as opposed to ester intermediate formation by L-haloacid dehalogenases. Through a docking simulation of DehE with D- and L-2-chloropropionic acid (D- and L-2CP), three binding residues namely W34, F37 and S188 were then identified. Additional investigation of DehE with non-degradable substrate, β-haloalkanoic acid (3CP) was carried out using molecular dynamics simulation. Interestingly, interconversion of the substrate moiety of 3CP nearby residue S188 has indicated highy unstable DehE-3CP complex with hydrogen bond distance range of 1.8 - 4.0 Å. In silico generated mutant S188V had achieved strong substrate interactions with a more stabilised 3CP maintaining a binding distance at ~2.0 Å. This was supported by an experimental work in which the activity of S188V towards 3CP was detected with a specific activity of 2.79 ± 0.01 µmol Cl–·min-1·mg-1 compared to zero activity for wild-type DehE. These details will develop rational design strategy to manipulate stereo-selectivity of dehalogenases for the future bioremediation and industrial applications. |
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| Physical Description: | xx, 205 leaves, : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 165-176) |