Crystal structure of ferric recombinant horseradish peroxidase

Abstract

Horseradish peroxidase (HRP), isolated from horseradish roots, is heavily glycosylated, making it difficult to crystallize. In this work, we produced recombinant HRP in E. coli and obtained an X-ray structure of the ferric enzyme at 1.63 Å resolution. The structure shows that the recombinant HRP contains four disulphide bonds and two calcium ions, which are highly conserved in class III peroxidase enzymes. The heme active site contains histidine residues at the proximal (His 170) and distal (His 42) positions, and an active site arginine (Arg 38). Surprisingly, an ethylene glycol molecule was identified in the active site, forming hydrogen bonds with His 42 and Arg 38 at the δ-heme edge. The high yields obtained from the recombinant expression system, and the successful crystallization of the enzyme pave the way for new structural studies in the future.

Keywords: Ferulic acid; Heme; Horseradish peroxidase; Peroxidase.

Fig. 1

A The UV–visible spectrum of recombinant ferric HRP (10 mM phosphate buffer, pH 7.0, 25° C). Addition of ferulic acid leads to no substantive change in the ferric spectrum. B Steady-state oxidation of guaiacol by recombinant HRP. The solid line shows the fit of the data to the Michaelis–Menten equation ([HRP] = 12 nM, 10 mM phosphate buffer, pH 7.0, 25 °C)

Fig. 2

The structure of recombinant ferric HRP. A The overall structure of HRP, showing the heme (in red), the proximal, and distal histidine residues and arginine residue (His 170, His 42, and Arg 38, respectively, in magenta) and ethylene glycol (EDO, green), calcium ions (olive green) and phosphate (orange). B The active site, showing the heme, ethylene glycol, and the active site residues. Colour scheme is as in (A); electron density (2F_o-F_c) map of the heme and ethylene glycol in the active site, contoured at 1.3σ. C Ethylene glycol forms hydrogen bonds with His 42 and Arg 38 (black dotted lines) and is at 4.0 Å from the heme iron (green dotted line). Colour scheme is the same as in (A). The figure was produced using PyMol [37]

Fig. 3

Comparison of binding locations in HRP. A Ethylene glycol (EDO, PDB ID 9H1M). B Ferulic acid (FA, PDB ID 6ATJ) [7]. C Benzhydroxamic acid (BHA, PDB ID 2ATJ) [22]. Colour scheme showing the heme (red), the Arg 38, and His 42 (magenta), and ethylene glycol, ferulic acid and benzhydroxamic acid (green). Water molecules are shown as blue spheres. The figure was produced using PyMol [37]. Superimposed images are shown in Figure S3