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 | |
发表期刊 | APPLIED AND ENVIRONMENTAL MICROBIOLOGY |
ISSN | 0099-2240 |
2020-07-24 | |
卷号 | 86期号:19页码:. |
关键词 | polychlorinated-biphenyls pcbs oxygenase component aerobic degradation dioxygenase metabolism b-356 regiospecificity biodegradation identification specificity |
研究领域 | Biotechnology & Applied Microbiology ; Microbiology |
DOI | 10.1128/AEM.01040-20 |
通讯作者 | Hu, Xiaoke(xkhu@yic.ac.cn) |
作者部门 | 海岸带生物学与生物资源保护实验室 |
英文摘要 | 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. |
文章类型 | Article |
资助机构 | National Natural Science Foundation of China ; Foreword Key Priority Research Program of the Chinese Academy of Sciences ; External Cooperation Program of the Chinese Academy of Sciences ; Key Research Project of Frontier Science of the Chinese Academy of Sciences |
收录类别 | SCI |
语种 | 英语 |
关键词[WOS] | polychlorinated-biphenyls pcbs ; oxygenase component ; aerobic degradation ; dioxygenase ; metabolism ; b-356 ; regiospecificity ; biodegradation ; identification ; specificity |
研究领域[WOS] | Biotechnology & Applied Microbiology ; Microbiology |
WOS记录号 | WOS:000577023300006 |
引用统计 | |
文献类型 | 期刊论文 |
条目标识符 | http://ir.yic.ac.cn/handle/133337/25280 |
专题 | 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 |
通讯作者 | Hu, XK |
作者单位 | 1.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 |
推荐引用方式 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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