In silico determination and analysis of Putative Haloalkanoic acid transport protein of Rhizobium Sp. Rc1

Abstract

There is a growing concern regarding the lack of an efficient solution to solve halogenated compound pollution in the environment. A Gram-negative bacterium, Rhizobium sp. RC1, which uses 2,2-DCP as one of its primary sources of carbon was previously isolated. However, the process of transporting haloacids into Rhizobium sp. RC1 has yet to be confirmed. A putative haloacid transport gene, dehrP, inside Rhizobium sp. RC1 is speculated to be responsible for this process. The aim of this research was to elucidate the function of this gene for the transport of haloacids into the cell. To achieve this, dehrP was initially analysed using several BLAST tools and then aligned using T-Coffee against other known transport proteins. The subsequent protein of this gene, DehrP, was concluded to belong in the Major Facilitator Superfamily (MFS) and Metabolite:H+ symporter (MHS) family of proteins. DehrP was determined to have nine transmembrane helices with MFS unique motifs between helices two and three, and helices eight and nine. Evolutionary analysis of DehrP was determined to have close relations to MHS family haloacid transporters, DehP, Deh4p and Dehp2 in Burkholderia caribensis MBA4. DehrP was modelled using Phyre2 with the transport protein XylE from Escherichia coli as the reference model. DehrP was compared with XylE in order to determine the proton and haloacid binding sites. The proton binding site of DehrP is made up of two residues, Asp36 and Arg130 whereas the assumed haloacid binding site residues are (Glu33, Trp34, Phe37, Arg75, Tyr271 and Ser402). To verify the assumption for the haloacid binding site, the binding site residues were replaced with alanine and the new sequence was named DehrPa. The 3D structures of DehrP and DehrPa were refined using 3Drefine in order to prepare them for docking simulations using AutoDock Vina. Docking simulations were done with four haloacids (2,2-DCP, MCA, D-2DCP and L-2DCP). The assumed substrate binding residues of DehrP was validated due to the lower binding affinity and lower binding accuracy of DehrPa. Unexpectedly, it was also found that 2,2-DCP was still able to bind to three other residues that was not mutated inside DehrPa. This study confirms haloacid binding site for DehrP of previous work with additional discovery of alternative binding residues specifically for 2,2-DCP.