Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119.

Yaprak Aslantas,Nur Basak Surmeli

doi:10.1155/2019/8080697

Abstract

Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.

Highlights

Enzymes catalyze reactions with high regioselectivity and enantioselectivity. ese key features of biocatalysts, i.e., enzymes, result in their increased utilization in the synthesis of pharmaceuticals and agrochemicals
Acid (ALA) for 16 hrs at 30°C. e N-His-CYP119 expression was induced with 0.5 mM Isopropyl ß-D-1thiogalactopyranoside (IPTG) and 0.3 mM ALA for 8 hrs at 30°C. e cells were harvested by centrifugation at 3800 rpm for 20 minutes. e cell pellet was kept at −80°C until purification
C-His-CYP119 and N-His-CYP119 were isolated with high yield and purity using immobilized metal affinity chromatography (IMAC). e UV-Visible spectra of WT CYP119 were in accordance to previous observations with Soret maximum absorbance at 414 nm and distinct α/β bands

Summary

Introduction

Enzymes catalyze reactions with high regioselectivity and enantioselectivity. ese key features of biocatalysts, i.e., enzymes, result in their increased utilization in the synthesis of pharmaceuticals and agrochemicals. Ese key features of biocatalysts, i.e., enzymes, result in their increased utilization in the synthesis of pharmaceuticals and agrochemicals. Heme-containing cytochrome P450 (P450) oxygenases are a attractive target because of their capability to catalyze oxidation of hydrocarbons with high efficiency and selectivity [1,2,3]. Erefore, the enzyme CYP119 from the acidothermophilic archaea Sulfolobus acidocaldarius is a very appealing target [7,8,9]. The native substrate for CYP119 is not known; CYP119 can catalyze epoxidation of styrene and the oxidation of N-acetyl-3,7dihydroxyphenoxazine (Amplex Red) by H2O2. One of the limitations in the practical application of CYP119 in the industry is that the expression and isolation of native CYP119 is tedious and time consuming [11]. One of the limitations in the practical application of CYP119 in the industry is that the expression and isolation of native CYP119 is tedious and time consuming [11]. ese problems can be solved by incorporation of polyhistidine tags (Histags) to facilitate isolation of the protein

Methods

Results

Discussion

Conclusion