The degradation of DDTs by Burkholderia xenovorans LB400 biphenyl dioxygenase and mutants

Abstract:

Dicholodiphenyltrichloroethanes (DDTs) is probably the best known and typical persistent organic pollutant in the world, which has been widely used in malaria control and agricultural deworming. They are still detected in various environmental matrices and has new input sources although their usage in agriculture has been banned in China and others. Numerous concerns have arisen over the past decades about the adverse environmental impacts(including harm to offshore ecosystem and human health) of DDTs. There has been a considerable interest over the last decades for the Rieske-type arylhydroxylating dioxygenases (RHDs) as they are seen as potentially capable of initiating their degradation. [Objective] In order to explore the degradation characteristics and mechanism of biphenyl dioxygenase(BPDO) on DDTs, we selected Burkholderia xenovorans LB400 biphenyl dioxygenase and its mutants to explore the degradation process of p,p'-DDT and o,p'-DDT. [Methods] Using BphAE_LB400 as parent, the mutant BphAE_S283M was obtained by two-step site-directed mutagenesis from Ser to Met. The degradation characteristics and mechanism of wild type and mutant were explored by comparing the catalytic performance of wild type and mutant to DDTs, simulating the structure of mutant protein and molecular docking. [Results] The data showed that BphAE_LB400 and BphAE_S283Mcould not be degraded p,p'-DDT, but BphAE_S283M metabolized o,p'-DDT and produced two stereoisomers. The structural analysis of BphAE_LB400 and BphAE_S283M showed that the reaction ring of p,p'-DDT did not coincide with the biphenyl reaction ring in the original crystal structure. In the o,p'-DDT-BphAE_S283M conformation, the proximal ring did not fit the biphenyl reactive ring as well, but its orientation toward the catalytic Fe²⁺ places two vicinal atoms at a distance(within 0.5 nm) that would allow a catalytic reaction. In addition, the surface area and volume of the catalytic cavity of BphAE_S283M is larger than that of BphAE_LB400, which is likely to contribute to the combination of BphAE_S283M and o,p'-DDT. [Conclusion] 283 is the key amino acid residue that affects the catalytic metabolism of BPDO to DDTs. It can affect the substrate specificity by adjusting the distance between the reaction carbon atom and the catalytic center and the size of the catalytic cavity. This will provide better insights about the bases for BPDO broad substrate range and about the mechanisms by which the enzyme evolves to change or expand its substrate range and its stereo- and regiospecificity.