Molecular Insight Into the Hydrolysis of Phthalate Esters by a Family IV Esterase

• Published in: Environmental microbiology Vol. 27; no. 7; pp. e70134 - n/a
• Main Authors: Rong, Zhen, Hong, Li‐Guo, Huo, Ying‐Yi, Li, Jixi, Zheng, Dao‐Qiong, Ha, Yang, Fan, Jeffrey, Xu, Xue‐Wei, Wu, Yue‐Hong
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.07.2025; Wiley Subscription Services, Inc
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biodegradation; Biodegradation, Environmental; Bioinformatics; Catalytic Domain; Chemical bonds; Contaminants; Crystal structure; Crystallography; Crystallography, X-Ray; DEHP; DEP; Diethyl phthalate; Dimerization; Dioctyl phthalate; Environmental cleanup; enzyme engineering; Enzymes; Esterase; Esterases; Esterases - chemistry; Esterases - genetics; Esterases - metabolism; Esters; Esters - metabolism; Ethylenediaminetetraacetic acids; family IV esterase; Hydrolysis; Metal ions; Microorganisms; Molecular docking; Molecular Docking Simulation; Mutagenesis; Mutagenesis, Site-Directed; Phthalate esters; Phthalates; Phthalic Acids - chemistry; Phthalic Acids - metabolism; Phylogenetics; Phylogeny; Steric hindrance; structure; Substrate Specificity; enzyme engineering; family IV esterase; DEHP; DEP; structure
• ISSN: 1462-2912; 1462-2920; 1462-2920
• DOI: 10.1111/1462-2920.70134

ABSTRACT Phthalate esters (PAEs) are prevalent environmental contaminants, with their biodegradation efficiently driven by microorganisms through ester bond hydrolysis. This study investigates the mechanism of Poc14, a novel family IV esterase, using x‐ray crystallography, bioinformatics, biochemistry and site‐directed mutagenesis. Phylogenetic analysis classifies Poc14 as a family IV esterase with conserved catalytic motifs crucial for its activity. Poc14 retains over 80% activity at 50°C for 4 h and tolerates up to 5% methanol or DMF, though surfactants like Tweens inhibit its function. Poc14 activity is independent of metal ions, and the addition of EDTA further enhances its activity by approximately 130%. The 1.8 Å crystal structure reveals a CAP domain and two substrate channels. Enzyme assays show Poc14 hydrolyses short‐chain diethyl phthalate (DEP) (Km = 0.068 mM, Vmax = 9975 μM/min/mg) but not long‐chain di(2‐ethylhexyl) phthalate (DEHP) due to steric hindrance. Molecular docking assessed Poc14's potential to hydrolyse DEP and DEHP after residue mutations, resulting in the Poc14‐AAG variant. Poc14‐AAG could hydrolyse one bond of DEHP and diester bonds of DEP. Our study positions Poc14 as a promising enzyme for environmental remediation, with potential for optimising DEHP degradation and exploring dimerisation effects. A family IV esterase, Poc14, was identified for degrading PAEs, with confirmed activity against DEP. Crystal structure analysis and molecular docking revealed key residues involved in PAE degradation. Mutations in these critical sites significantly enhanced DEP hydrolysis and partially degraded DEHP, highlighting its potential for pollutant remediation.