Genomic characterization and in silico analysis of dehalogenases from Mesorhizobium loti strain tono

• Main Author: Zakary, Sefatullah
• Format: Thesis
• Language: English
• Published: 2021
• Subjects: Q Science (General)
• Online Access: http://eprints.utm.my/id/eprint/101829/1/SefatullahZakaryMFS2021.pdf.pdf; http://eprints.utm.my/id/eprint/101829/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:146358

Halogenated compounds are extensively utilized in different industrial applications such as pesticides and herbicides and cause severe environmental problems because of their toxicity and persistence. Degradation of these compounds by the biological method is a significant method to reduce these recalcitrant. Mesorhizobium loti is important for nitrogen fixation in legume roots. Up to now, no report has indicated Mesorhizobium loti can produce dehalogenase enzymes. Thus, a total of twenty-five genomes of Mesorhizobium loti strains from the National Center for Biotechnology Information (NCBI) were analyzed. These strains notably carry dehalogenase genes and were further investigated. The relative ratio of haloalkane and haloacid dehalogenase type II or L-type from all twenty-five genomes was 26% and 74%, respectively, suggesting type II dehalogenase is common. Surprisingly, only Mesorhizobium loti strain TONO carries four dehalogenases and therefore it was further characterized. The chromosome of Mesorhizobium loti strain TONO contains four haloacid dehalogenase type II genes namely, dehLt1 (MLTONO_2099), dehLt2 (MLTONO_3660), dehLt3 (MLTONO_4143), and dehLt4 (MLTONO_6945), and their corresponding enzymes were designated as DehLt1, DehLt2, DehLt3, and DehLt4, respectively. The only haloalkane dehalogenase gene (MLTONO_4828) was located upstream of the dehLt3 gene and its amino acid share 88% identity with DmlA of Mesorhizobium japonicum MAFF 303099. The putative haloacid permease gene designated as dehrPt (MLTONO_0284) was located downstream of the dehLt1 and its amino acids show 69% identity with haloacid permease of Rhizobium sp. RC1. The gene encoding helix-turn-helix (HTH) motif family DNA-binding protein regulator and LysR family transcriptional regulator genes were also identified, possibly for regulatory functions. The type II dehalogenase, DehLt4 possess high sequence identity (48.18% and 42.73%) with the well-established DehIVa and L-DEX, respectively. Thus, in the current study, an in silico approach was used for homology modelling and docking assessment of newly identified DehLt4, type II dehalogenase to predict its ability to degrade selected haloalkanoic acids and haloacetate. The study aimed to establish the catalytic tendencies of the enzymes to optimally degrade the selected halogenated haloacids. The refined modelled structure of DehLt4 using GROMACS 5.1.2 software revealed satisfactory scores of ERRAT (94.73%), Verify3D (90.83%) and PROCHECK (99.05 %) assessments. Active site prediction by blind docking, and multiple sequence alignment indicated the catalytic triads for DehLt4 were Asp9-Lys149-Asn175. Both L-2-chloropropionic acid (L-2CP) and trichloroacetate (TCA) docked with DehLt4 exhibited binding energy of -3.9 kcal/mol. While the binding energy for D-2-chloropropionic acid (D-2CP) and monochloroacetate (MCA) was -3.8 kcal/mol and -3.1 kcal/mol, respectively. Thus, the findings of the study successfully identified the catalytic important residues of DehLt4 for possible pollutant degradation. The genomic studies as such, have good potential to be screened for new type of dehalogenases based on basic molecular structure and functions analysis.