Biochemical Characterization of Arylamine N- acetyltransferases From Vibrio vulnificus

Xinning Liu; Yuanchang Liu; Guangjian Zhao; Yidan Zhang; Lu Liu; Juan Wang; Yifan Wang; Siyu Zhang; Xin Li; Dongliang Guo; Peng Wang; Ximing Xu

doi:10.3389/fmicb.2020.595083

Biochemical Characterization of Arylamine N- acetyltransferases From Vibrio vulnificus

Front Microbiol. 2021 Jan 18:11:595083. doi: 10.3389/fmicb.2020.595083. eCollection 2020.

Authors

Xinning Liu^{1

2

3}, Yuanchang Liu⁴, Guangjian Zhao¹, Yidan Zhang¹, Lu Liu¹, Juan Wang¹, Yifan Wang⁵, Siyu Zhang⁶, Xin Li⁶, Dongliang Guo⁷, Peng Wang⁵, Ximing Xu^{1

2

3}

Affiliations

¹ Marine Drug Screening and Evaluation Platform (QNLM), School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
² Center for Innovation Marine Drug Screening & Evaluation, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China.
³ Institute of Bioinformatics and Medical Engineering, Jiangsu University of Technology, Changzhou, China.
⁴ Quality Control Department, Qilu Children's Hospital of Shandong University, Jinan, China.
⁵ College of Food Science and Engineering, Ocean University of China, Qingdao, China.
⁶ School of Life Sciences, Lanzhou University, Lanzhou, China.
⁷ School of Information Science and Engineering, Yanshan University, Qinhuangdao, China.

Abstract

Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H₂O₂. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn²⁺ and Cu²⁺. The kinetic parameters K _m and V _max were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the T _m , T _agg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.

Keywords: Arylamine N-acetyltransferases; Vibrio vulnificus; acetyl coenzyme A; acetylation; enzyme.