Research paper Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa Amanda Bernardes a, 1 , Larissa C. Textor a, 1 , Jademilson C. Santos a, 1 , Nazaret Hidalgo Cuadrado b, 2 , Eduard Ya. Kostetsky c , Manuel G. Roig b , Vassiliy N. Bavro e , Jo ~ ao R.C. Muniz a , Valery L. Shnyrov d , Igor Polikarpov a, * a Instituto de Física de S~ ao Carlos, Universidade de S~ ao Paulo, Av. Trabalhador S~ aocarlense 400, S~ ao Carlos, SP 13560-970, Brazil b Departamento de Química Física, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain c Departament of Biochemistry, Microbiology and Biotechnology, Far Eastern Federal University, 690600 Vladivostok, Russia d Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, 37007 Salamanca, Spain e Institute of Microbiology and Infection, University of Birmingham, Birmingham B152TT, United Kingdom article info Article history: Received 29 July 2014 Accepted 25 January 2015 Available online 7 February 2015 Keywords: Plant peroxidase Chamaerops excelsa Oxidoreductases Protein crystallography Protein oligomerization abstract Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural el- ements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage. © 2015 Published by Elsevier B.V. 1. Introduction Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidore- ductase) are heme proteins that catalyze the oxidoreduction of a broad variety of peroxides. Most commonly, peroxidases catalyze the oxidation of organic substrates, while reducing H 2 O 2 to water. This process involves multiple-reactions and a number of intermediate enzyme forms, and is known as the PouloseKraut mechanism, which plays a key role in several metabolic responses of all peroxidases [1]. Peroxidases are important for many biological responses and processes, such as defense against pathogenic mi- croorganisms, cell wall formation, and lignification [2]. The peroxidase superfamily includes animal and non-animal peroxidases, and the latter are generally subdivided into three classes, all sharing a similar three-dimensional fold despite their low amino acid sequence identity [3,4]. Class I includes intracellular enzymes, such as plant ascorbate peroxidases, yeast cytochrome c peroxidases and bacterial catalases. Class II is composed of secreted peroxidases encoded exclusively by fungal organisms, including lignin peroxidase and Mn 2þ -dependent peroxidases. Finally, Class III consists of secreted plant peroxidases with molecular weights * Corresponding author. E-mail address: ipolikarpov@ifsc.usp.br (I. Polikarpov). 1 These authors contributed equally to the work. 2 Present address: Instituto de Estudios Biofuncionales, Departamento de Quí- mica-Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi http://dx.doi.org/10.1016/j.biochi.2015.01.014 0300-9084/© 2015 Published by Elsevier B.V. Biochimie 111 (2015) 58e69