Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93
IntroductionCPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-01-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1081094/full |
| _version_ | 1797945032023474176 |
|---|---|
| author | Yang Li Yang Li Zhen Rong Zhengyang Li Henglin Cui Jixi Li Xue-Wei Xu |
| author_facet | Yang Li Yang Li Zhen Rong Zhengyang Li Henglin Cui Jixi Li Xue-Wei Xu |
| author_sort | Yang Li |
| collection | DOAJ |
| description | IntroductionCPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis.MethodsX-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p-NP (p-nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates.ResultsThree core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p-NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC.DiscussionThe study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases. |
| first_indexed | 2024-04-10T20:48:57Z |
| format | Article |
| id | doaj.art-422706ccbb3440029c67c5230942e4cd |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-10T20:48:57Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-422706ccbb3440029c67c5230942e4cd2023-01-23T18:46:51ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-01-011310.3389/fmicb.2022.10810941081094Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93Yang Li0Yang Li1Zhen Rong2Zhengyang Li3Henglin Cui4Jixi Li5Xue-Wei Xu6School of Oceanography, Zhejiang University, Zhoushan, ChinaKey Laboratory of Marine Ecosystem Dynamics, Ministry of Natural Resources and Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou, ChinaKey Laboratory of Marine Ecosystem Dynamics, Ministry of Natural Resources and Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, ChinaSchool of Food and Biological Engineering, Jiangsu University, Zhenjiang, ChinaState Key Laboratory of Genetic Engineering, School of Life Sciences, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, ChinaKey Laboratory of Marine Ecosystem Dynamics, Ministry of Natural Resources and Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou, ChinaIntroductionCPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis.MethodsX-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p-NP (p-nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates.ResultsThree core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p-NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC.DiscussionThe study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1081094/fullmarine bacterial carboxylesterasecrystal structureenzyme catalysisprodrugp-nitrophenylsubstrate specificity |
| spellingShingle | Yang Li Yang Li Zhen Rong Zhengyang Li Henglin Cui Jixi Li Xue-Wei Xu Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 Frontiers in Microbiology marine bacterial carboxylesterase crystal structure enzyme catalysis prodrug p-nitrophenyl substrate specificity |
| title | Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
| title_full | Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
| title_fullStr | Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
| title_full_unstemmed | Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
| title_short | Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
| title_sort | structural insights into catalytical capability for cpt11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase e93 |
| topic | marine bacterial carboxylesterase crystal structure enzyme catalysis prodrug p-nitrophenyl substrate specificity |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1081094/full |
| work_keys_str_mv | AT yangli structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT yangli structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT zhenrong structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT zhengyangli structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT henglincui structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT jixili structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT xueweixu structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 |