Molecular Dynamics Simulations of BcZBP,A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity Vasiliki E. Fadouloglou, †,‡,⊥ Athanassios Stavrakoudis, § Vassilis Bouriotis, ‡,| Michael Kokkinidis, ‡,| and Nicholas M. Glykos* ,† Department of Molecular Biology and Genetics, Democritus UniVersity of Thrace, UniVersity campus, GR-68100, Alexandroupolis, Greece, Department of Biology, UniVersity of Crete, PO Box 2208, GR-71409, Heraklion, Crete, Greece, Department of Economics, UniVersity of Ioannina, GR-45110, Ioannina, Greece, and Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110, Heraklion, Crete, Greece Received May 11, 2009 Abstract: BcZBP is an LmbE-like, homohexameric, zinc-dependent deacetylase from the opportunistic pathogen Bacillus cereus with three, thus far uncharacterized, homologues in B. anthracis. Although its specific substrate is still unknown, the enzyme has been shown to preferentially deacetylate N-acetylglucosamine and diacetylchitobiose via an active site based on a zinc-binding motif of the type HXDDX n H. In the crystal structure, the active site is located at a deep and partially blocked cleft formed at the interface between monomers related by the molecular 3-fold axis, although the major, in structural terms, building block of the enzyme is not the trimer, but the intertwined dimer. Here, we report results from a 50 ns molecular dynamics simulation of BcZBP in explicit solvent with full electrostatics and show that (i) the view of the intertwined dimer as the major structural and functional building block of this class of hexameric enzymes is possibly an oversimplification of the rather complex dynamics observed in the simulation, (ii) the most mobile (with respect to their atomic fluctuations) parts of the structure coincide with three surface loops surrounding the active site, and (iii) these mobile loops define the active site’s accessibility, and may be implicated in the determination of the enzyme’s specificity. 1. Introduction Bacillus anthracis has recently attracted significant interest, mainly because of its putative usage as a biological weapon. 1 Part of this interest was subsequently transferred to more benign, but still closely related to B. anthracis, species like B. cereus, an opportunistic bacterium causing food poison- ing. 2 In a drive to characterize, both functionally and structurally, deacetylases that are shared between these two organisms (and which may be implicated in metabolic pathways of biotechnological and pharmaceutical interest), we have recently reported the characterization, purification, crystallization and crystal structure determination of BcZBP, a homohexameric, LmbE-like, zinc-dependent deacetylase from B. cereus. 3-5 BcZBP is the product of the bc1534 gene, with three, thus far uncharacterized, homologues in B. anthracis sharing sequence identities (at the protein level) of 96%, 28%, and 24%, respectively. Functional studies 5 * To whom correspondence should be addressed. Tel. +302551030620. Fax. +302551030620, glykos@mbg.duth.gr. URL: http://www.mbg.duth.gr/~glykos/. † Democritus University of Thrace. ‡ University of Crete. § University of Ioannina. | Institute of Molecular Biology and Biotechnology (IMBB). ⊥ Present address: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA, Cambridge, England (UK). J. Chem. Theory Comput. 2009, 5, 3299–3311 3299 10.1021/ct9002338 CCC: $40.75  2009 American Chemical Society Published on Web 11/04/2009