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Engineering Burkholderia xenovorans LB400 BphA through site directed mutagenesis at site 283

Li, Junde, Min, Jun, Wang, Yuan, Chen, Weiwei, Kong, Yachao, Guo, Tianyu, Mahto, Jai Krishna, Sylvestre, Michel et Hu, Xiaoke (2020). Engineering Burkholderia xenovorans LB400 BphA through site directed mutagenesis at site 283 Applied and Environmental Microbiology , vol. 86 , nº 19. p. 1-17. DOI: 10.1128/AEM.01040-20.

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Résumé

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO) catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of broader substrate range, BphAE(S283M,) BphAE(p4-S283M) and BphAE(RR41-S283M) variants were created from parent enzymes BphAE(LB400,) BphAE(p4) and BphAE(RR41), respectively by substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the k (cat)/K(m) value of BphAE(S283M,) BphAE(p4-S283M) and BphAE(RR41-S283M) significantly increased compared to their parent enzyme. Meanwhile, we determined the Steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-CB, 2,2',6,6'-CB and 2,3',4,4',5-CB. We compared the catalytic reaction of BphAE(LB400) and its variants toward 2,2'-CB, 2,5-CB and 2,6-CB. The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site, thereby its site of hydroxylation. And this was confirmed by docking experiments. We also assessed the substrates range of BphAE(LB400) and its variants with degradation activity. BphAE(S283M) and BphAE(p4-S283M) were clearly improved in oxidizing some of the 3-6 chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. It will certainly be meaningful elements for understanding the effect of the residue corresponding to 283 in other Rieske oxygenase enzymes.ImportanceThe segment 280-283 in BphAEs is located at the entrance of the catalytic pocket and it shows variation conformation. In previous works, the results have been suggested but never proved residue Ser283 of BphAE(LB400) might play a role in substrate specificity. In the present paper, we found the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3',4,4'-CB, 2,2',6,6'-CB and 2,3',4,4',5-CB. Meanwhile, the Ser283Met substitution altered regiospecificity of BphAE toward 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAE(S283M) and BphAE(p4-S283M) were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3-6 Chlorines; Table 4), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, this study provides better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

Type de document: Article
Informations complémentaires: document 01040-20
Mots-clés libres: Burkholderia xenovorans LB400; biphenyl dioxygenase; directed evolution; enzyme catalysis; enzyme mutation; polychlorinated biphenyls; regiospecificity; substrate range
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 20 juill. 2021 03:59
Dernière modification: 20 juill. 2021 03:59
URI: https://espace.inrs.ca/id/eprint/11625

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