Development of a novel catalytic amyloid displaying a metal- dependent ATPase-like activity Octavio Monasterio, Esteban Nova, Rodrigo Diaz-Espinoza * Laboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile article info Article history: Received 19 November 2016 Accepted 2 December 2016 Available online 3 December 2016 Keywords: Amyloid Peptide Catalysis Hydrolysis ATP Manganese abstract Amyloids are protein aggregates of highly regular structure that are involved in diverse pathologies such as Alzheimer's and Parkinson's disease. Recent evidence has shown that under certain conditions, small peptides can self-assemble into amyloids that exhibit catalytic reactivity towards certain compounds. Here we report a novel peptide with a sequence derived from the active site of RNA polymerase that displays hydrolytic activity towards ATP. The catalytic reaction proceeds in the presence of the divalent metal manganese and the products are ADP and AMP. The kinetic data shows a substrate-dependent saturation of the activity with a maximum rate achieved at around 1 mM ATP. At higher ATP concen- trations, we also observed substrate inhibition of the activity. The self-assembly of the peptide into amyloids is strictly metal-dependent and required for the catalysis. Our results show that aspartate- containing amyloids can also be catalysts under conditions that include interactions with metals. Moreover, we show for the first time an amyloid that exerts reactivity towards a biologically essential molecule. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Amyloids are highly ordered protein aggregates that have been classically associated to relevant pathologies such as Alzheimer's, Parkinson's and Prion diseases among others [1]. Their overall structural architecture is characterized by a cross-beta fold that grows along a central axis to form large filaments [2,3]. The involvement of amyloids in several pathologies has labeled them as toxic particles, although no universal mechanism for toxicity has yet been established [4]. In recent years however, increasing evi- dence of non-toxic functional amyloids has been reported [5]. Their unique mechanical and structural features can be exploited by nature to carry novel functions such as biofilm formation, tran- scriptional regulation, hormone storage, etc. [6e8]. These works have also led to propose amyloids as novel bio-nanomaterials that can have diverse biotechnological applications [9]. The diverse properties of amyloids have recently been expanded with the discovery of catalytic reactivity emerging from small self- assembled peptides [10,11]. These rationally designed catalytic amyloids exhibited potent esterase activity towards the model compound p-nitrophenylacetate when histidine residues were alternated with hydrophobic groups in the peptide sequences [10]. The solvent-exposed histidines were shown to coordinate zinc ions (Zn þ2 ) to form catalytically active amyloid-metal complexes that showed enzymatic-like features from the kinetic data. In a more recent report, different histidine-containing peptides displaying similar esterase activity emerged from a random peptide library using a central amyloid-prone motif [11]. These findings can have important implications that include not only a putative involve- ment of amyloid catalytic reactivity in cellular toxicity but also a plausible role in molecular evolution. In fact, amyloids have been proposed as potentially relevant prebiotic entities due to their unique physical properties such as spontaneous self-assembly of their peptide constituents and high stability against environmental changes including temperature and pH, among others [12]. The discovery of catalytic reactivity emerging from amyloids may imply that small prebiotic peptides can in principle harbor novel func- tionalities. Interestingly, mixtures of several amino acids consid- ered prebiotic have recently been shown to yield peptides that spontaneously self-assembled into amyloid structures, providing a proof of principle to this idea [13]. In the present study, we report the design and development of a novel catalytic amyloid that displayed hydrolytic activity towards Abbreviations: Th-T, Thioflavin-T; Mg þ2 , magnesium ion; Mn þ2 , manganese ion; ATP, adenosine triphosphate. * Corresponding author. Las Palmeras 3425, Santiago, CP7800003, Chile. E-mail address: rodrigo.diaz.e@u.uchile.cl (R. Diaz-Espinoza). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.12.011 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 482 (2017) 1194e1200