Biochemical Characterization of Arylamine N-acetyltransferases From Vibrio vulnificus.

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

doi:10.3389/fmicb.2020.595083

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 Km and Vmax were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the Tm, Tagg 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.

Highlights

Vibrio vulnificus (V. vulnificus) is a ubiquitous gram-negative aquatic bacterium that belongs to the Vibrionaceae family
The results of the (VIBVN)NAT sequence alignment showed that the C-terminal region of (VIBVN)NAT is approximately 20 amino acids shorter than the C-terminal region of other kinds of NATs (Figure 1)
The activity of (VIBVN)NAT was tested by the PNPA and dithiobis-(2-nitrobenzoic acid) (DTNB) methods with different acetyl donors and acceptors

Summary

Introduction

Vibrio vulnificus (V. vulnificus) is a ubiquitous gram-negative aquatic bacterium that belongs to the Vibrionaceae family. V. vulnificus can generally invade human hosts through two routes: oral consumption and wound infection. Oral consumption of seafood such as, primarily, raw oysters or raw molluscan shellfish, can cause severe gastroenteritis or septicemia infection (Kim et al, 2015). Wound infection is generally acquired by exposure to contaminated seawater or seafood products, resulting in necrotizing fasciitis (Huang et al, 2016). The incidence of this infection has increased dramatically worldwide because of the spreading geographical distribution of V. vulnificus infection, and the disease was found even in some previously unaffected regions (Vezzulli et al, 2013). The mortality rate of infection will dramatically increase from 33 to 100% in less than 48 h (Klontz et al, 1988). Understanding the resistance mechanism of V. vulnificus is urgently needed

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Conclusion