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abstracts
Biphenyl dioxygenase(BphAE) & biphenyl dehydrogenase(BphB) catalyze first two steps of aerobic degradation of biphenyl & various polychlorinated biphenyls,PCBs. These enzymes are of interest for their potential to oxidize toxic pollutants & manufacture of fine chemicals. We have determined the crystal structures of two variants of BphAE from B. xenovorans LB400, viz BphAERR41 & BphAEp4, evolved by site directed mutagenesis. We have compared the structures of wild type BphAELB400 & BphAEp4. Biochemical properties of BphAELB400 variants with single substitutions, T335A or F336M shows that residue 336 contacts biphenyl & influences regiospecificity of the reaction, but do not enhance enzyme's reactivity toward 2,6-dichlorobiphenyl. However, residue 335 does not contact biphenyl but contributes significantly to expand enzyme's substrate range. Crystal structure of variant BphAERR41 explains the transformation of dibenzofuran & 2-chlorodibenzofuran. This study provides structural insight for expanded substrate range of Rieske-type oxygenases through mutations that enhance the plasticity and/or mobility of enzyme segments lining the catalytic site. Enzyme, BphB catalyze 2nd step of PCB degradation pathway. We have determined the crystal structure of apoenzyme, binary complex with NAD+ & ternary complexes with NAD+2,3-dihydroxybiphenyl from P. pnomenusa B356. A crystal structure representing an intermediate state of the enzyme was also obtained where the substrate binding loop was ordered in comparison with apo & binary forms but was displaced significantly w.r.t ternary structure. These structures reveal that the substrate binding loop is highly mobile & changes conformation during ligand binding, starting from a disorganized loop in apo state to a well organized loop in ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate wide range of substrates. For the 1st time, a combination of structural, biochemical, & molecular docking studies of BphBB356 elucidate the unique ability of enzyme to transform the cis-dihydrodiols of double meta-, para-, & ortho-substituted chlorobiphenyls.
abstracts
Candidatus Liberibacter asiaticus is a fastidious prokaryotic α-proteobacterium that has not been cultured as yet. It is the causative agent of Citrus Huanglongbing (HLB) is a disease that infects citrus plants causing huge losses to the citrus industry. The plants employ a variety of defense mechanisms to combat the pathogen attack which mainly involves the formation of reactive oxygen species (ROS) mainly hydrogen peroxide, peroxynitrite and organic hydroperoxides. The microbe on the other hand has antioxidant proteins that counter the reactive oxygen species. Peroxiredoxin (Prx) which is known to play a crucial role in peroxide detoxification is a super family of one of such antioxidant proteins. In this work, we have determined the crystal structure of Bacterioferritin Comigratory Protein (BCP) that belongs to 1-Cys Prx enzyme having peroxidatic/sulfenic acid cysteine (C-47) but lacks the resolving cysteine. Furthermore, we are investigating structural aspects to gain insight into the binding of different peroxide substrates at the active site. Site directed mutagenesis has been done to introduce the non-conserved resolving cysteine to study its effect on peroxidase activity in both wild type and mutant form. Biochemically, both BCP 1-cys and BCP 2-cys have shown peroxidase activity. DNA protection activity against oxidative damage and cell-line based assays has implicated its protective role towards the hypoxic condition of cells. Hence, the protein becomes a lucrative target for ligands which might be potential antimicrobials.
