Extraction, cloning and characterization of a novel 4-hydroxyphenylpyruvate dioxigenase enzyme from paenarthrobacter nitroguajacolicus with the ability to produce arene oxide

Ahmadpur, Nanasrin; Mohamadzadeh, Soraya; Jafarian, Vahab

doi:10.22034/cmr.2024.8364.3292

Extraction, cloning and characterization of a novel 4-hydroxyphenylpyruvate dioxigenase enzyme from paenarthrobacter nitroguajacolicus with the ability to produce arene oxide

Document Type : Research Paper

Authors

nanasrin Ahmadpur ¹

soraya mohamadzadeh ²

Vahab Jafarian ³

¹ Master&rsquo;s Degree in Biochemistry, Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran.

² Master Degree in Biochemistry, Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran.

³ Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

10.22034/cmr.2024.8364.3292

Abstract

4-hydroxyphenylpyruvate dioxygenase is an iron-dependent homotetrameric enzyme. In this study, we extracted the gene encoding this enzyme from Paenarthrobacter nitroguajacolicus strain Znu06. The recombinant enzyme was cloned into the pET28a vector. Then expressed in E. coli BL21(DE3) using IPTG inducer and purified using a Ni-NTA Agarose column. SDS-PAGE was employed to evaluate the expression level and the Bradford assay was used to determine the concentration. The activity of the enzyme was assessed at different temperatures and substrate concentrations, followed by performance evaluation using HPLC and examining reaction products. The structure of the enzyme and interactions were studied using bioinformatics tools and servers. The PCR results confirmed a genomic fragment with a length of 1020 bp which encoded an enzyme with 339 amino acid residues with a molecular weight of 34 KD. The sequence was deposited in NCBI with the code MN197545.1. The activity of the enzyme was confirmed through biochemical analysis using spectrophotometry and fluorometry. Sequence alignment using ESPript3 confirmed highly conserved residues, including His 152, His‌ 216, Glu 267, Tyr 257, His 199, and His 248 in the enzyme's active sites. The enzyme seems to promote the reaction towards aryl oxide production due to the absence of hydrogen bond formation in the active site, leading to the lack of homogentisic acid production.

Keywords

Arene oxide

Bioinformatics

Cloning

Fluorescence

4-hydroxyphenylpyruvate dioxygenase

Subjects