title: Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli.
abstract: 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 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/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)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). 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.
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dempsey
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Bond
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Carpenter
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Santiago
authors.initials: S
authors.lastname: Rodriguez Ospina
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA. Electronic address: merkler@usf.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Merkler
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D051379:Mice / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C037817:benzoyl-coenzyme A; D000217:Acyltransferases; C020680:glycine acyltransferase; D005998:Glycine
keywords: Benzoyl-CoA; Escherichia coli; Glycine N-acyltransferase; Hippurate; N-Acylglycine; Steady-state kinetics