Cargando…
Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93
INTRODUCTION: CPT11 (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-[...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9901791/ https://www.ncbi.nlm.nih.gov/pubmed/36756200 http://dx.doi.org/10.3389/fmicb.2022.1081094 |
_version_ | 1784883097061294080 |
---|---|
author | Li, Yang Rong, Zhen Li, Zhengyang Cui, Henglin Li, Jixi Xu, Xue-Wei |
author_facet | Li, Yang Rong, Zhen Li, Zhengyang Cui, Henglin Li, Jixi Xu, Xue-Wei |
author_sort | Li, Yang |
collection | PubMed |
description | INTRODUCTION: CPT11 (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. METHODS: X-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. RESULTS: Three 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. DISCUSSION: The 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. |
format | Online Article Text |
id | pubmed-9901791 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-99017912023-02-07 Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 Li, Yang Rong, Zhen Li, Zhengyang Cui, Henglin Li, Jixi Xu, Xue-Wei Front Microbiol Microbiology INTRODUCTION: CPT11 (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. METHODS: X-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. RESULTS: Three 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. DISCUSSION: The 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. Frontiers Media S.A. 2023-01-11 /pmc/articles/PMC9901791/ /pubmed/36756200 http://dx.doi.org/10.3389/fmicb.2022.1081094 Text en Copyright © 2023 Li, Rong, Li, Cui, Li and Xu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Li, Yang Rong, Zhen Li, Zhengyang Cui, Henglin Li, Jixi Xu, Xue-Wei Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 |
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 | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9901791/ https://www.ncbi.nlm.nih.gov/pubmed/36756200 http://dx.doi.org/10.3389/fmicb.2022.1081094 |
work_keys_str_mv | AT liyang structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT rongzhen structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT lizhengyang structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT cuihenglin structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT lijixi structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 AT xuxuewei structuralinsightsintocatalyticalcapabilityforcpt11hydrolysisandsubstratespecificityofanovelmarinemicrobialcarboxylesterasee93 |