The ten hotspots at positions 49, 51, 58, 230, 232, 248, 251, 257, 260 and 262 form a welldefined cluster with interesting structural features. All of them are located in the b-strand that forms a "wall" of the de novo active site. Therefore, many of them could be considered as "active site" residues, except for the fact that in all cases the side chains are pointing in the opposite direction, i.e., away from the active site (Figure ). Chemical shift perturbation of these residues upon binding of transition-state analogue may simply reflect small changes in the shape of the de novo active site. For this reason, mutations at these hotspot positions, even if they do not involve active-site side chains, may plausibly alter the active-site shape and in doing so affect catalysis.