(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Alphaproteobacteria: NE > Sphingomonadales: NE > Erythrobacteraceae: NE > Altericroceibacterium: NE > Altericroceibacterium indicum: NE
Molecular evidence
Database
No mutation 1 structure: 7X8L: Microbial family VII carboxylesterase E93 Wild-type No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MARTRYGPVIGKVEQGALAFKGIPYGAPTSGSGRFMPPTPPQPWSTPLRA FDYGPTAPQSDPQDALESGAADARESEDCLTLNVWTPSLNDQRKRPVMVW LHGGGLWRLSAAGDYQAGTHLAAHSDVVMVSPNHRLNVLAHAYLDEYDPA FAGSSSAGMLDLVLALKWVRDNIEEFGGDPDNVTIFGQSGGGQKVSFLMA MPAAAGLFHKAIIQSGPAPLALEKPYARELSARLLTLLDIPKNRVRDIQN VPLDAIMRAYYQIFEELGGFGVMGVIQDFAPVVDDVALPQHPFWNGASPL SRDVPLMIGCTRTEMTEYFLASNPGAAKRDFAAVTAQLEPVFGMQAPAVV AHYRATHPTASPWEVDALIRSDWPTRLFTQRIADEQVKLGGAPVWMYRMD WQTTARDGLLMSPHAIDIPFVLDTVGTEPVEPGQLAEQQRMMQQMNNAWV SFARNGNPQNKYIPPWQPYNSTSRPTMIFNLHSHMANDPDGSDLAFLKKD LANLEVVAGGVTHPPVLEKSS
Reference
Title: Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93 Li Y, Rong Z, Li Z, Cui H, Li J, Xu XW Ref: Front Microbiol, 13:1081094, 2023 : PubMed
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.
Altericroceibacterium indicum Carboxylic ester hydrolase E93
