Conformational Dynamics of Active Site Loop in Escherichia coli Phytase Amol V. Shivange, 1,2 Ulrich Schwaneberg, 2 Danilo Roccatano 1 1 School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, Bremen D-28759, Germany 2 Department of Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen D-52074, Germany Received 9 June 2010; accepted 9 June 2010 Published online 22 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21513 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION P hytases (myo-inositol hexakis phosphohydrolase), catalyze the release of phosphate by stepwise hydro- lysis of phosphomonoester bonds in phytate (myo- inositol-1,2,3,4,5,6-hexakisphosphate). Phytate is the major storage form of phosphate and inositol, predominantly occurring in cereal grains, legumes, and oilseeds. 1 However, the phosphorous in this form is essen- tially unavailable to monogastric animals such as human beings, dogs, birds, swine, and poultry due to lower or negli- gible phytase activity. 2 Furthermore, phytate is considered to be anti-nutrient because of its polyanionic chelating property that forms complexes with several divalent cations of major nutritional importance like Zn 21 , Cu 21 , Ca 21 , Mn 21 Fe 21 , and Mg 21 . Phytate can also form complexes with proteins and amino acids. 3 Undigested phytate leads to decrease in bioavailability of inorganic phosphate (Pi) and nutritionally important elements to the animals and as a result undi- gested/unabsorbed phytate is excreted as large amounts of Pi into the environment resulting in environmental pollution. 4 For proper skeletal growth, these animals need Pi at suitable concentration, thus phytases are considered of a significant value in upgrading the nutritional quality of phytate rich feed, supplementation of phytase to the feed additive provided an alternative to tackle both these conditions effectively. In addition to its major application in the animal nutri- tion, phytases are also used in human nutrition, inhibitory effect of inositol hexaphosphate (phytate) as well as penta- phosphate on iron absorption has been reported, 5 causing the high prevalence of iron deficiency in infants from devel- oping countries, women of fertile age, or vegetarians. The Conformational Dynamics of Active Site Loop in Escherichia coli Phytase Additional Supporting Information may be found in the online version of this article. Correspondence to: Danilo Roccatano; e-mail: d.roccatano@jacobs-university.de ABSTRACT: Phytases catalyze the release of phosphate by stepwise hydrolysis of phytate, a major source of phosphate in cereal grains, legumes, and oilseeds. Phytase improves, as a feed supplement, the nutritional quality of phytate rich diets and eventually reduce environmental pollution. Recently, phytases from enterobacteriaceae family have attracted industrial interest due to their high specific activity (2500–4000 U/mg). However, only limited information is available concerning structural dynamics of this class of enzymes. In this study, 50 nanosecond molecular dynamics simulation was performed on two Escherichia coli phytase structures (closed and open active site loop) to investigate conformational dynamics of the active site loop. Cluster analysis and principal component analysis (PCA) reveal significant difference in the conformational dynamics of active site compared to reported crystal structure. Molecular dynamic studies indicated that the movement in the active site of E. coli phytase is mainly confined by the active site loop resulted in wider opening of the loop in absence of phytate. The molecular dynamics studies highlight the possible role of loop residues as prerequisite for highly active phytases. # 2010 Wiley Periodicals, Inc. Biopolymers 93: 994–1002, 2010. Keywords: phytase; molecular dynamics simulations; principal component analysis; cluster analysis; active site loop; domainselect analysis V V C 2010 Wiley Periodicals, Inc. 994 Biopolymers Volume 93 / Number 11