Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93

• Published in: Frontiers in microbiology Vol. 13; p. 1081094
• Main Authors: Li, Yang, Rong, Zhen, Li, Zhengyang, Cui, Henglin, Li, Jixi, Xu, Xue-Wei
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 11.01.2023
• Subjects: crystal structure; enzyme catalysis; marine bacterial carboxylesterase; Microbiology; p-nitrophenyl; prodrug; substrate specificity; prodrug; crystal structure; enzyme catalysis; marine bacterial carboxylesterase; p-nitrophenyl; substrate specificity
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2022.1081094

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. X-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with -NP ( -nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates. Three core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated 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 -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. 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.