Molecular Dynamics of Psychrophilic Enzyme Mutants Figure 4. Anisotropic temperature factors computed for fluctuations in the essential subspace. The anisotropic temperature factors are shown as grey ellipsoids centred on the Ca atoms of the average structures from simulations. Grey spheres represent the coordinated ions. The regions of loop 7 and 8 are highlighted by circles in AHA and PPA, respectively. doi:10.1371/journal.pone.0024214.g004 common elements for a-amylase fold, allowing the correct orientation of the nucleophile side chain in the catalytic site. A group of salt bridges (group IV, Table S1 in Supporting Information S1) and their networks, similar to that of PPA, and differing from those of AHA, can be identified in the mutants (Figure 7, 8, Table S1 in Supporting Information S1) and their relevance will be discussed in details in the next sections. This confirms a trend toward mesophilic-like properties in AHA mutants, as well as long range effects induced by the mutations. The selected mutations cannot restore the overall electrostatic networks of the mesophilic amylase, since several salt bridges of PPA lack suitable corresponding residues in AHA and the opposite (groups II and III, Table S1 in Supporting Information S1). In particular, several arginine residues of PPA are absent in AHA (Table S1 in Supporting Information S1), accounting for differential salt-bridges interactions and networks, in line with the stabilizing effects induced by replacement of AHA lysines with homoarginines [54]. Modification in dynamics properties around L7/L8 loop Domain A is the central and largest domain of a-amylases; it accommodates the active site at the heart of its (b/a)8 barrel structure. ba loops forming the substrate-binding cleft (Figure 1), have a role in substrate recognition and processing [55–57] in several a-amylases. In particular, loop L8 has been identified as an initial contact point for the incoming substrate, mediating enzy- matic specificity [58], whereas loop L7 may rearrange upon substrate binding and release, contributing to the substrate placement inside the active site [59]. The entity of L7 flexibility is decreased in most of the mutant variants (Figure 3 and 4), accompanied by a concomitant disappearance of one of the highest correlated networks PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24214