J1 acylase, a glutaryl‐7‐aminocephalosporanic acid acylase from <i>Bacillus laterosporus</i> J1, is a member of the α/β‐hydrolase fold superfamily

Yau, Ming-Hon; Wang, Jun; Tsang, Paul Wai-Kei; Fong, Wing‐Ping

doi:10.1016/j.febslet.2006.01.069

Cited by 9 publications

(4 citation statements)

References 27 publications

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“…Moreover, cefepime degradation products by α/β hydrolase and other enzymes, e.g., β-lactamase, were identified (Figures S9–S11, Table S5). The resistance mechanism has a double-edged sword effect, as it decreases the antibiotic selection pressure to the surrounding sensitive species, alleviating AMR development in microbial communities …”

Section: Resultsmentioning

confidence: 99%

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics

Wang

Feng

Lü

2022

Environ. Sci. Technol.

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Antibiotics exert selective pressures on clinically relevant antibiotic resistance. It is critical to understand how antibiotic resistance evolves in environmental microbes exposed to subinhibitory concentrations of antibiotics and whether evolutionary dynamics and emergence of resistance are predictable. In this study, Comamonas testosteroni isolated from wastewater activated sludge were subcultured in a medium containing 10 ng/mL cefepime for 40 days (∼300 generations). Stepwise mutations were accumulated, leading to an ultimate 200-fold increase in the minimum inhibitory concentration (MIC) of cefepime. Early stage mutation in DNA polymerase-encoding gene dnaE2 played an important role in antibiotic resistance evolution. Diverse resistance mechanisms were employed and validated experimentally, including increased efflux, biofilm formation, reduced antibiotic uptake, and drug inactivation. The cefepime minimal selective concentrations (MSCs) and relative fitness of susceptible, intermediate, and resistant mutants were determined. Agent-based modeling of the modified Moran process enabled simulations of resistance evolution and predictions of the emergence time and frequency of resistant mutants. The unraveled cefepime resistance mechanisms could be employed by broader bacteria, and the newly developed model is applicable to the predictions of general resistance evolution. The improved knowledge facilitates the assessment, prediction, and mitigation of antibiotic resistance progression in antibiotic-polluted environments.

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Section: Resultsmentioning

confidence: 99%

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics

Wang

Feng

Lü

2022

Environ. Sci. Technol.

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“…The results suggested the presence of conserved residues in DthA which are characteristic of ␣/␤ hydrolases and which form part of a Ser-His-Asp catalytic triad in previously characterized proteins (9). Residues S166 and D292 from DthA aligned with the serine and aspartic acid residues that have been identified, respectively, as the nucleophile and the acidic residues that form part of the catalytic triad in the homologous proteins (2,7,10,19). No histidine residues from DthA aligned with the catalytic histidine residues in the Clustal X alignment.…”

mentioning

confidence: 95%

Bacterial Degradation of N,N -Diethyl- m -Toluamide (DEET): Cloning and Heterologous Expression of DEET Hydrolase

Rivera-Cancel¹,

Bocioaga²

2007

Appl Environ Microbiol

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Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.N, N-diethyl-m-toluamide (DEET) is the active ingredient in most topical insect repellent products. Approximately 30% of the U.S. population use DEET-containing products, and domestic usage of DEET is estimated to be 1800 tonnes annually (16). It has been frequently detected in U.S. streams (in 74% of the streams surveyed) in the low parts-per-billion levels (5).There is very little information about the microbial metabolism of DEET. Only partial degradation by the fungi Cunninghamella elegans and Mucor ramannianus R-56, via N oxidation and N deethylation, has been shown previously (13). Here we report the isolation of a bacterium capable of utilizing DEET as a sole carbon and energy source. We also describe the identification and heterologous expression of a gene from this bacterium encoding a DEET hydrolase. To our knowledge, this is the first report of a microorganism able to use DEET as a sole source of carbon and energy.Chemicals. DEET (98%), 3-methylbenzoate (99%), 3-methylcatechol (99%), diethylamine (Ͼ99%), benzenesulfonyl chloride (99%), acetaldehyde (99.5%), and glacial acetic acid were purchased from Acros Organics (Morris Plains, NJ). Phenylmethylsulfonyl fluoride (PMSF) and aprotinin were obtained from Sigma (St. Louis, MO) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) from Fluka (Buchs, Switzerland). Methanol and sodium nitroprusside were purchased from Fisher Scientific (Pittsburgh, PA).Isolation of Pseudomonas putida DTB. Activated sludge from a municipal wastewater treatment plant in Ithaca, NY, was enriched with DEET (2.6 mM) according to standard protocols (4). A bacterial strain was isolated in pure culture and designated DTB. A fragment of the 16S rRNA gene from strain DTB was amplified by PCR and sequenced using the universal primers 27F and 1492R (6). The sequence of this fragment was compared with those deposited in the GenBank database using BLAST (1) and was found to be 100% identical to that of the 16S rRNA gene from Pseudomonas putida KT2440 over 1,419 nucleotides.Pathway of DEET degradation by P. putida DTB. To determine the DEET degradation pathway, DTB was inoculated into minimal salts medium (MSM) (4) amended with 2.6 mM DEET. Growth was monitored by measuring attenuance at 600 nm. The culture was sampled over a 98-h period after inoculation. Samples were diluted with 1 volume of methanol and centrifuged at 21,000 ϫ g for 10 min. The supernatants were analyzed by high-performance liquid chromatography (HPLC) (8) by monitoring absorbance at 220 nm and compared with DEET, 3-methylbenzoate, and 3-methylcatechol standards. The mobile phase consisted of 60% methanol and 40% 40 mM acetic acid.HPLC...

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“…cocaine esterase (RhCocE) [31] and J1 glutaryl 7-ACA acylase (J1) [32]. Both the RhCocE and the J1 enzyme do not require an α -amino group in the substrate (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

Blum

Bommarius

2010

Journal of Molecular Catalysis B: Enzymatic

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α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of kcat of 72.5 s−1 and KM of 1.1 mM. The synthesis parameters α, βo, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, βo = 42.8 M−1, and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.

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J1 acylase, a glutaryl‐7‐aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the α/β‐hydrolase fold superfamily

Cited by 9 publications

References 27 publications

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics

Bacterial Degradation of N,N -Diethyl- m -Toluamide (DEET): Cloning and Heterologous Expression of DEET Hydrolase

Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

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