Purificação, caracterização e avaliação da atividade anti-helmíntica de uma peroxidase do látex de Calotropis procera (Ait.)R. Br

Abstract

Latex is a complex fluid enriched in hydrolytic enzymes, such as proteases and chitinases; however, the occurrence and role of peroxidases in this material remain poorly understood. Peroxidases (EC 1.11.1.7) are widely distributed across biological systems due to their involvement in numerous physiological processes. These enzymes are classified as heme and non-heme peroxidases, with heme-containing peroxidases accounting for approximately 84% of all described peroxidases, according to the RedOxiBase database. Among them, class III plant peroxidases constitute a large multigene family of heme-dependent enzymes that share a similar three-dimensional structure and a common catalytic mechanism for hydrogen peroxide degradation. They are involved in the metabolism of reactive oxygen species (ROS), hormone synthesis and degradation, fruit growth, defense responses, and cell wall synthesis and maintenance. In this context, we report the purification and structural characterization of an atypical peroxidase (CpLP-POX) isolated from the latex of C. procera. Initial analyses revealed a high-molecular-weight enzyme (~112 kDa) that dissociated into ~35 kDa subunits under denaturing conditions, suggesting a trimeric organization. Structural modeling corroborated the homotrimeric assembly of the protein. The CpLP-POX monomers exhibited a conserved catalytic triad (Arg–His–Asn), four disulfide bonds, and the overall fold characteristic of class III plant peroxidases. The enzyme displayed maximal activity at pH 6.0 and 40 °C and was inhibited by EDTA, sodium azide, and thiol-reducing agents, consistent with the profile of Ca2+-associated heme-dependent peroxidases. CpLP-POX did not exhibit inhibitory effects on nematode egg hatching, suggesting that its physiological role may be more closely related to oxidative defense or protection against microbial invasion. Collectively, these findings provide the first biochemical and structural evidence of a trimeric peroxidase in plants, expanding the known structural diversity of this enzyme class and contributing to a deeper understanding of its biological functions.