Enzyme and Microbial Technology 51 (2012) 35–39 Contents lists available at SciVerse ScienceDirect Enzyme and Microbial Technology jou rn al h om epage: www.elsevier.com/locate/emt Prediction of substrate-binding site and elucidation of catalytic residue of a phytase from Bacillus sp. Ahmed Abass Osman 1 , Peram Ravindra Babu 1 , Kamarthapu Venu, Khareedu Venkateswara Rao, Vudem Dashavantha Reddy ∗ Centre for Plant Molecular Biology, Osmania University, Hyderabad 500007, AP, India a r t i c l e i n f o Article history: Received 17 November 2011 Received in revised form 28 March 2012 Accepted 29 March 2012 Keywords: Bacillus phytase Site-directed mutagenesis Phytate Catalytic site a b s t r a c t The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed - propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme. © 2012 Elsevier Inc. All rights reserved. 1. Introduction In cereals, legumes and oil seeds, phytate/phytic acid {myo- inositol(1,2,3,4,5,6)hexakisphosphate} is the principal storage form of organic phosphorus (P) and myo-inositol(Ins). Cycling of P in soil is intimately associated with the activity of soil microor- ganisms [1,2]. Phytic acid, a polyanionic chelating agent, forms complexes with divalent cations, such as Fe 2+ , Ca 2+ , Mg 2+ , etc., and proteins, resulting in the anti-nutritional activity. Phytases catalyse the hydrolysis of phytate to less phosphorylated Ins phosphates, yielding one molecule of inositol and six molecules of inorganic phosphate [3]. Phytases are identified from a number of sources including microorganisms, plants and animals. Earlier, we have iso- lated a thermostable phytase encoding gene from a Bacillus species and expressed in Escherichia coli. Based on the aminoacid sequence similarity and catalytic activity, the enzyme was characterized as a -propeller phytase (BPP) [4]. Kerovuo et al. [5] reported that the Bacillus PhyC gene- encoded phytase, with 3- and 6-phytase activities, upon sequential ∗ Corresponding author. Tel.: +91 40 27098087; fax: +91 40 27096170. E-mail address: vdreddycpmb@yahoo.com (V.D. Reddy). 1 These authors contributed equally to this work. hydrolysis of alternate phosphates of the phytate, yielded Ins(2,4,6)triphosphate and Ins(1,3,5)triphosphate. Phytases of B. subtilis and B. amyloliquefaciens exhibited 3-phytase activity and yielded Ins(2,4,6)triphosphate as an end product [6]. Site- specific mutagenesis of B. amyloliquefaciens DS11 phytase revealed aminoacid residues Y159, E211, E260 and D314 essential for bind- ing of calcium ions, and K76 and R122 for binding of phytate [7]. A synthetic beta-propeller phytase, with higher thermostability and activity over a broad range pH, was designed by structure-guided consensus approach using aminoacid sequences from Bacillus phy- tases. The synthetic phytase disclosed 99% similarity with the phytase crystal structure of B. amyloliquefaciens [8]. The crystal structure of B. subtilis phytase complexed with calcium ions and substrate analogue inositol hexasulfate (IHS) was solved at 1.25 ˚ A resolution [9]. Crystal structures of partially/fully loaded Ca 2+ states of phytase (2POO) revealed the presence of two high affinity cal- cium binding sites which contributed to increased thermostability. Also, three additional low affinity Ca 2+ binding sites were found involved in turning on the catalytic activity [10]. Shin et al. proposed catalytic mechanism (s) for thermostable phytase using crystallog- raphy, site-directed mutagenesis and biochemical studies [11]. In view of different mechanisms and catalytic activities of var- ious -propeller phytases, the present study has been carried out to identify the probable substrate-binding site and elucidation of 0141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2012.03.010