Dataset Information

Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase.

ABSTRACT: Many sites are often co-contaminated with multiple pesticides. To date, there are no reports on simultaneous degradation of different classes of pesticides by a natural microorganism. In this work, we aim at constructing a live biocatalyst able to simultaneously hydrolyze carbaryl and chlorpyrifos. For this purpose, carbaryl hydrolase (CH) was displayed on the cell surface of a chlorpyrifos-degrading bacterium Stenotrophomonas sp. strain YC-1 using N- and C-terminal domain of ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. The localization of INPNC-CH fusion protein in the outer membrane fraction was demonstrated by cell fractionation followed by Western blot analysis. Surface display of INPNC-CH was further confirmed by proteinase accessibility experiment and immunofluorescence microscope. CH was present in an active form on cell surface without causing any growth inhibition, suggesting that the INP-based display system is a useful tool for surface expression of macromolecular heterologous proteins on the bacterial cell surface. Because surface-displayed CH has free access to pesticides, this bacterium can be used as a whole-cell biocatalyst for efficient hydrolysis of pesticides.

SUBMITTER: Yang C

PROVIDER: S-EPMC5645314 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase.

Yang Chao C Xu Xiaoqing X Liu Yanping Y Jiang Hong H Wu Yunbo Y Xu Ping P Liu Ruihua R

Scientific reports 20171017 1

Many sites are often co-contaminated with multiple pesticides. To date, there are no reports on simultaneous degradation of different classes of pesticides by a natural microorganism. In this work, we aim at constructing a live biocatalyst able to simultaneously hydrolyze carbaryl and chlorpyrifos. For this purpose, carbaryl hydrolase (CH) was displayed on the cell surface of a chlorpyrifos-degrading bacterium Stenotrophomonas sp. strain YC-1 using N- and C-terminal domain of ice nucleation prot ...[more]

PMID: 29042673

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Project description:Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCH?(Tmd)a were found to be monomeric with molecular mass of ?68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 ?M and 69 to 73 ?mol · min-1 · mg-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

Project description:AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

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Dataset Information

Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase.

Publications

Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase.

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