International Journal of Biological Macromolecules 94 (2017) 378–385 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsevier.com/locate/ijbiomac A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803 Tripti Pandey 1 , Rohit Shukla 1 , Harish Shukla, Amit Sonkar, Timir Tripathi ∗ , Arvind Kumar Singh ∗ Molecular & Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong, 793022, Meghalaya, India a r t i c l e i n f o Article history: Received 7 September 2016 Received in revised form 13 October 2016 Accepted 14 October 2016 Available online 17 October 2016 Keywords: Synechocystis Glutathione S-transferase Glutathione Peroxide Peroxidase Modeling Molecular dynamic simulation a b s t r a c t Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S- transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109 M) and high Vmax (0.663 mol min −1 ) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Peroxides and hydroxyl radicals are highly reactive and bio- logically toxic chemical molecules. Even though peroxides are not free radicals, they participate in redox reactions. A comparison of peroxides with dioxygen suggests that peroxides are a more reac- tive oxygen species [1]. While colliding with organic molecules, peroxides may extract an electron from it; this electron renders the propagation of a chain reaction, leading to the oxidation of bio-molecules such as membrane lipids, proteins, and DNA [2]. Unlike superoxides, hydrogen peroxide (H 2 O 2 ) and cumenehy- droperoxide (CuOOH) are highly diffusible through membranes and aqueous compartments and can directly inactivate sensitive Abbreviations: GST, glutathione S-transferase; GSH, reduced glutathione; CuOOH, cumene hydrogen peroxide; Non-Se-GPX, non-selenium dependent glu- tathione peroxidase; RMSD, root mean square deviation; RMSF, root mean square fluctuation; ED, essential dynamics; PCA, principal component analysis. ∗ Corresponding authors. E-mail addresses: tripathi@gmail.com (T. Tripathi), aksingh nehu@yahoo.co (A.K. Singh). 1 Both the authors contributed equally to the work. enzymes at a low concentration [3]. Similar to superoxides, per- oxides are stable and, therefore, are less toxic than other reactive oxygen species. However, they provide the environment for the generation of highly reactive hydroxyl free radicals inside the cells [4]. These highly reactive hydrogen free radicals in oxygen-derived species are known to be involved in cell injury or death. Cells catalyze the detoxification process of peroxide radi- cals using various enzymes including peroxidase, catalase, and glutathione peroxidases. Some reports have suggested that glu- tathione s-transferases (GSTs) also show peroxidase activity in the presence of reduced glutathione (GSH) [5–9]. GSTs are enzymes that play an important role in the phase II of cellular detoxifica- tion. They catalyze the conjugation of the thiol group of GSH to electrophilic compounds resulting in soluble products that can be removed from the cytosol by ATP dependent GS–X pumps. More- over, a growing number of non–detoxification functions have now been attributed to GSTs [10,11]. Three–dimensional structures of several GSTs have been solved, including complexes with substrate- GSH, substrate analogues, and products. Structurally, most GSTs exist in dimeric state though monomeric [12,13] and a tetrameric GST [14,15] has been reported. Despite the low level of sequence identity across the classes, all http://dx.doi.org/10.1016/j.ijbiomac.2016.10.040 0141-8130/© 2016 Elsevier B.V. All rights reserved.