کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020492 | 1542344 | 2014 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34 kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5 mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app = (5.2 ± 0.20) Ã 102 Mâ1 sâ1, and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app = (4.5 ± 0.27) Ã 105 Mâ1 sâ1. Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 97, May 2014, Pages 23-28
Journal: Protein Expression and Purification - Volume 97, May 2014, Pages 23-28
نویسندگان
Daniel R. Dempsey, Jason D. Bond, Anne-Marie Carpenter, Santiago Rodriguez Ospina, David J. Merkler,