Dataset Information

Folate-Dependent Hydrolysis of Acetyl-Coenzyme A by Recombinant Human and Rodent Arylamine N-Acetyltransferases.

ABSTRACT: Arylamine N-acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis-Menten kinetic constants (Vmax and Km) of the folate-dependent AcCoA hydrolysis for human NAT1/NAT2, and the rodent analogs rat Nat1/Nat2, mouse Nat1/Nat2, and hamster Nat1/Nat2. We also compared apparent Vmax values for AcCoA hydrolysis and N-acetylation of the substrate para-aminobenzoic acid (PABA). Human NAT1 and its rodent analogs rat Nat2, mouse Nat2 and hamster Nat2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Rates of AcCoA hydrolysis were between 0.25 - 1% of the rates for N-acetylation of PABA catalyzed by human NAT1 and its rodent orthologs. In contrast to human NAT1, human NAT2 and its rodent analogs rat Nat1, mouse Nat1, and hamster Nat1 did not hydrolyze AcCoA in a folate-dependent manner. These results are consistent with the possibility that human NAT1 and its rodent analogs regulate endogenous AcCoA levels.

SUBMITTER: Stepp MW

PROVIDER: S-EPMC4545580 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Publications

Folate-Dependent Hydrolysis of Acetyl-Coenzyme A by Recombinant Human and Rodent Arylamine N-Acetyltransferases.

Stepp Marcus W MW Mamaliga Galina G Doll Mark A MA States J Christopher JC Hein David W DW

Biochemistry and biophysics reports 20150901

Arylamine <i>N</i>-acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis-Menten kinetic constants (Vma ...[more]

PMID: 26309907

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Project description:Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

Project description:BACKGROUND:2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway. RESULT:In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66?phzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66?phzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe3+ to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3?phzB?lon?pykA?rsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date. CONCLUSION:In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.

Dataset Information

Folate-Dependent Hydrolysis of Acetyl-Coenzyme A by Recombinant Human and Rodent Arylamine N-Acetyltransferases.

Publications

Folate-Dependent Hydrolysis of Acetyl-Coenzyme A by Recombinant Human and Rodent Arylamine N-Acetyltransferases.

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