Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283
Li, JD; Min, J; Wang, Y; Chen, WW; Kong, YC; Guo, TY; Mahto, JK; Sylvestre, M; Hu, XK1,2,4
Source PublicationAPPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN0099-2240
2020-07-24
Volume86Issue:19Pages:.
Keywordpolychlorinated-biphenyls pcbs oxygenase component aerobic degradation dioxygenase metabolism b-356 regiospecificity biodegradation identification specificity
MOST Discipline CatalogueBiotechnology & Applied Microbiology ; Microbiology
DOI10.1128/AEM.01040-20
Department海岸带生物学与生物资源保护实验室
Abstract

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 a broader substrate range, the BphAE(S283M), BphAE(p4-S283M), and BphAE(RR41-S283M) variants were created from the parent enzymes BphAE(LB400), BphAE(p4), and BphAE(RR41), respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the k(cat)/K-m values of BphAE(S283M), BphAE(pR-S283M), and BphAE(RR41-S283M )were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-tetrachlorobiphenyl (2,3',4,4'-CB), 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-CB), and 2,3',4,4',5-pentachlorobiphenyl (2,3',4,4',5-CB). We compared the catalytic reactions of BphAE(LB)(400) and its variants toward 2,2-'dichlorobiphenyl (2,2'-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges 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. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes. IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAE(LB400) might play a role in substrate specificity. In the present paper, we found that 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 the 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 to 6 chlorines), 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, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

SubtypeArticle
Indexed BySCI
Language英语
WOS Keywordpolychlorinated-biphenyls pcbs ; oxygenase component ; aerobic degradation ; dioxygenase ; metabolism ; b-356 ; regiospecificity ; biodegradation ; identification ; specificity
WOS Research AreaBiotechnology & Applied Microbiology ; Microbiology
WOS IDWOS:000577023300006
Citation statistics
Document Type期刊论文
Identifierhttp://ir.yic.ac.cn/handle/133337/25280
Collection海岸带生物学与生物资源利用重点实验室_海岸带生物学与生物资源保护实验室
Corresponding AuthorHu, XK
Affiliation1.Chinese Acad Sci, Yantai Inst Coastal Zone Res, Key Lab Coastal Biol & Bioresource Utilizat, Yantai, Peoples R China;
2.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Peoples R China;
3.Univ Chinese Acad Sci, Beijing, Peoples R China;
4.Chinese Acad Sci, Ctr Ocean Megasci, Qingdao, Peoples R China;
5.Indian Inst Technol Roorkee, Dept Biotechnol, Roorkee, Uttarakhand, India;
6.Inst Natl Rech Sci INRS, Inst Armand Frappier, Laval, PQ, Canada
Recommended Citation
GB/T 7714
Li, JD,Min, J,Wang, Y,et al. Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2020,86(19):..
APA Li, JD.,Min, J.,Wang, Y.,Chen, WW.,Kong, YC.,...&Hu, XK.(2020).Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,86(19),..
MLA Li, JD,et al."Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283".APPLIED AND ENVIRONMENTAL MICROBIOLOGY 86.19(2020):..
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