Identification of L‐Asparagine as a Novel Endogenous Substrate for Human Arylamine N‐Acetyltransferase 1
Authors: Carlisle Samantha M. , Trainor Patrick J. , Hein David W. 
Published: November 2023

INTRODUCTION

Human arylamine N‐acetyltransferase 1 (NAT1) is classically described as phase II xenobiotic metabolizing enzyme that catalyzes the N‐acetylation of arylamine and hydrazine substrates using acetyl‐coenzyme A (acetyl‐CoA) as the acetyl donor. NAT1 expression varies inter‐individually based on epigenetic modifications as well as single nucleotide polymorphisms (SNPs) although the effects of all SNPs are not well understood. Increased expression of NAT1 has been associated with several cancers, including estrogen receptor positive (ER+) breast cancer. Although NAT1 is expressed in most tissues and found in nearly every species an endogenous function has yet to be elucidated.

OBJECTIVE

To identify endogenous substrate(s) for human NAT1.

METHODS

To interrogate metabolic differences associated with varying levels of NAT1 in breast cancer, we performed a global untargeted metabolomics study on MDA‐MB‐231 breast cancer cells expressing parental (Scrambled), increased (Up), decreased (Down, Crispr 2–12), and knockout (Crispr 2–19, Crispr 5–50) levels of NAT1. All cell lines used as samples were constructed via siRNA or CRISPR‐Cas9 technology from the MDA‐MB‐231 cell line purchased from ATCC®. We identified one metabolite, N‐acetylasparagine, whose abundance distribution among the sample groups suggested it was the product of N‐acetylation by NAT1. To verify that NAT1 can catalyze the N‐acetylation of L‐asparagine we performed a spectrophotometric enzymatic assay using human NAT1 recombinantly expressed in yeast.

RESULTS

The mean of the scaled relative abundance of N‐acetylasparagine was increased approximately 3.0‐fold in the Up cell line, and decreased approximately 2.3, 2.5, 11.6, and 17.9‐fold in the Down, Crispr 2–12, Crispr 2–19, and Crispr 5–50 cell lines respectively, compared to the Scrambled (parental activity) cell line. Pairwise comparisons of abundance distribution to the Scrambled cell line were each significant (multiplicity adjusted p<0.0002). Of all metabolites detected, N‐acetylasparagine was the only metabolite to follow this abundance distribution pattern. The spectrophotometric assay confirmed the N‐acetylation of L‐asparagine by recombinant human NAT1 but not recombinant human NAT2.

CONCLUSIONS

Our results indicate that L‐asparagine can be N‐acetylated by NAT1 using acetyl‐CoA as the acetyl donor, albeit at much lower levels than the prototypic NAT1 substrate 4‐aminobenzoic acid (PABA). NAT2, an isozyme of NAT1 with more limited tissue distribution, was not shown to catalyze the N‐acetylation of L‐asparagine. To further validate our findings, we will develop a more sensitive N‐acetylation assay using high performance liquid chromatography (HPLC) methods to detect and quantify N‐acetylasparagine and L‐asparagine. Recently it has been postulated that L‐asparagine acts as an amino acid exchange factor and this exchange with extracellular amino acids regulates mTOR complex 1 activation, nucleotide synthesis, and cancer cell proliferation. These data suggest NAT1 may be playing a role in cancer through differences in the rates of N‐acetylation of L‐asparagine.

Support or Funding Information

This study was partially supported by USPHS grant T32‐ES011564.