Family Report for: HNLyase_Bact

Carboxylesterase PytH, isolated from a pyrethroid degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin and bifenthrin. To elucidate the catalytic mechanism of PytH, here we report the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported alpha/beta-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230 and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrates binding; both lid and core domains are involved in substrate binding and the lid domain induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household, however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. It showed that it belongs to the alpha/beta-hydrolase fold proteins with typical catalytic Ser-His-Asp triad though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket endowed PytH binding bigger pyrethroids family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deeper the understanding of alpha/beta-hydrolase members.

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many alpha/beta-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.

The long-term and excessive use of pyrethroid pesticides poses substantial health risks and ecosystem concerns. Several bacteria and fungi have been reported that could degrade pyrethroids. The ester-bond hydrolysis using hydrolases is the initial regulatory metabolic reaction of pyrethroids. However, the thoroughly biochemical characterization of hydrolases involved in this process is limited. Here, a novel carboxylesterase, designated as EstGS1 that could hydrolyze pyrethroid pesticides was characterized. EstGS1 showed low sequence identity (<27.03%) compared to other reported pyrethroid hydrolases and belonged to the hydroxynitrile lyase family that preferred short short-chain acyl esters (C2 to C8). EstGS1 displayed the maximal activity of 213.38 U/mg at 60 degreesC and pH 8.5 using pNPC2 as substrate, with K(m) and V(max) were 2.21 +/- 0.72 mM and 212.90 +/- 41.78 microM/min, respectively. EstGS1 is a halotolerant esterase and remains stable in 5.1 M NaCl. Based on molecular docking and mutational analysis, the catalytic triad of S(74)-D(181)-H(212) and three other substrate-binding residues I(108), S(159), and G(75) are critical for the enzymatic activity of EstGS1. Additionally, 61 and 40 mg/L of deltamethrin and lambda-cyhalothrin were hydrolyzed by 20 U of EstGS1 in 4 h. This work presents the first report on a pyrethroid pesticide hydrolase characterized from a halophilic actinobacteria.

Carboxylesterase PytH, isolated from a pyrethroid degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin and bifenthrin. To elucidate the catalytic mechanism of PytH, here we report the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported alpha/beta-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230 and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrates binding; both lid and core domains are involved in substrate binding and the lid domain induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household, however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. It showed that it belongs to the alpha/beta-hydrolase fold proteins with typical catalytic Ser-His-Asp triad though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket endowed PytH binding bigger pyrethroids family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deeper the understanding of alpha/beta-hydrolase members.

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many alpha/beta-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.