These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

# 1999 International Union of Crystallography Printed in Denmark ± all rights reserved 2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1.2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was puri®ed by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sittingdrop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 A Ê on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 A Ê .