The mutation of Lys300 to a glutamine results in an active but chloride-independent enzyme (Table 1), thus mimicking the residue organization found in Cl−-independent bacterial α-amylases such as those from Bacillus amyloliquefaciens and B. licheniformis (Machius et al. 1998). The absence of chloride in the Lys300Gln mutant structure is clearly seen from the difference Fourier density maps calculated on the basis of phases from the native enzyme with chloride included (Fig. 4). Compared to the three-dimensional structure of the native enzyme, the side-chain of Gln300 now replaces a water molecule (Wat1001), being the third water molecule (when starting counting from the active site) in the chloride water pocket described earlier (Aghajari et al. 1998a). Furthermore, Arg172 turns its guanidinium group in another fashion than the one found in the chloride binding site in the native enzyme (Fig. 2), toward the active site, where it is located between Asp174 and Glu200. In addition to these changes, Wat1003 (first water molecule in the chloride water pocket) has moved into the site where chloride was previously bound, and very interestingly, the putative catalytic water molecule, Wat1004 (Aghajari et al. 1998a,b), has disappeared. In other α-amylase three-dimensional structures, this conserved water molecule is in a perfect position to attack the α-1,4 glycosidic bond in the substrate. Both aspects, Wat1003 replacing the Cl− ion as well as the absence of Wat1004, are probably correlated with the eightfold lower activity of this mutant compared with the native enzyme (Table 1). One could argue that the missing Wat1004 is just not visible in the electron density because of the rather poor data (Table 3), but in this particular case, which concerns a water molecule that is found to be conserved in all α-amylase three-dimensional structures, in which it is firmly hydrogen bonded to the catalytic residues Glu200 and Asp264, it obviously does not seem fortuitous. It should be noticed that this inactive enzyme is quite difficult to crystallize and that the few crystals obtained are very small and unstable. This indicates that this mutant enzyme probably is quite labile.
When comparing with the corresponding site in α-amylase from B. licheniformis (BLA; Machius et al. 1998), which is constituted of Arg229 (Arg172, AHA), Asn326 (Asn262, AHA), and Gln360 (Gln300, AHA), it is seen that although these residues are identical in nature, the sites are quite different spatially. The Asn (262, AHA; 326, BLA) side-chains are the only residues which display exactly the same conformation in the two enzymes, whereas the arginine (172, AHA; 229, BLA) side-chains adopt diverging positions in the respective enzymes. Considering a sphere with a radius of ∼20 A˚ centered on the chloride binding site, it appears that a significant number of residues, present in the first and second shell, are not conserved. Among the most important differences, we find that in the B. licheniformis enzyme, Phe284 (Thr221, AHA) and Trp41 (Gln33, AHA), as well as the proton donor in the hydrolytic reaction, Glu261 (Glu200 AHA), point directly into the binding site. Within this sphere, the number of aromatic residues in BLA is higher than in the psychrophilic amylase, and this site is clearly more positively charged in AHA than in BLA, even with the mutation Lys300Gln. In addition, the water molecule, which seems to have moved from the water pocket (Aghajari et al. 1998a), Wat1003, to the chloride binding site is found in BLA as well and corresponds to Wat2007.