Structural characterization of Lepidium draba peroxidase using computational and experimental studies

Peroxidases have wide applications in many different areas such as medical diagnostics and industry. Recently we cloned a new kind of peroxidase gene from Lepidium draba (LDP) in pET28a(+) vector and its expression was optimized in prokaryotic expression system and its sequence was also determined. Pairwise sequence alignment using Clustal omega program demonstrated that LDP had 93% and 88.96% sequence similarity and identity with horseradish peroxidas C1A (HRP C1A), respectively. However, characterization of secondary and tertiary structure of LDP was the main objective of current study. Therefor Raptor X web server at http://raptorx.uchicago.edu/ was used for prediction of the secondary structure of LDP. Furthermore, far-UV circular dichroism (CD) was used to determine the secondary structure of recombinant LDP which produced in prokaryotic hosts. Ligands and binding sites were predicted at http://www.sbg.bio.imperial.ac.uk/. SiteHound-web and MetaPocket were employed in order to predict the interactive residues. According to the results of Raptor X algorithm, secondary structure components of LDP were 52% coil, 46% helix, and 1.95% beta sheet. CD results showed that LDP consists of 43.6% α-helix, 11.9% β-sheet, 15.3% β-turn, and 29.2% random coil. Based on the results, the helix content in LDP is near to the helix content in the class III plant peroxidases, which is approximately 35–40%. The binding site of LDP comprised of Met28, Leu37, Arg38, Phe41, His 42, Pro69, Ser73, Pro139, Ala140, Pro141, Phe152, Leu163, Leu166, Ser167, Gly169, His170, Asn175, Phe179, Phe221, Ileu244, Thr246, and Phe277. Totally, the results demonstrated that LDP is a homologue of HRP C1A and its folding is done correctly in prokaryotic systems.