Histidine acid phytases (HAPhy) are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA), homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal “RHGXRXP” and C-terminal “HD.” Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 “SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF” was present in 38 protein sequences representing clusters 1 (PhyA) and 2 (PhyB). Cluster 3 (AppA) contains motif 9 “KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP” as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. This in silico analysis might contribute in the classification and future genetic engineering of this most diverse class of phytase. 1. Introduction Phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate; IP6) is the major storage form of phosphorus (P), representing approximately 80% of P in soil [1], 65–80% of total P in grains [2], and up to 80% of P in manures from monogastric animals [3]. Phytate exists primarily as metal phytate complex with nutritionally important cations, that is, Ca2+, Fe2+, and Zn2+ [4]. Phytases (IP6 phosphohydrolase) are a class of phosphatases which catalyses hydrolysis of phytate to inositol phosphates, inorganic phosphorus, and myo-inositol [5], also lowers down affinity of phytate to associated minerals and proteins [6], and thus increases bioavailability of P, minerals, and proteins for growth and development of plants and animals [7–9]. Phytases are widely distributed among plants [10, 11], certain animal tissues, and microbial cells [12–15]. To date, four classes of phytases have been characterized in terrestrial organisms: histidine acid phytase (HAPhy), cysteine phytase (CPhy), purple acid phosphatase (PAP), and -propeller phytase (BPPhy) [16, 17]. HAPhys are the most studied and diverse class of
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