Several mutants of KAU have been structurally investigated in order to rationalize their enzymatic behavior and to establish the role of the mutated residues in the building and functioning of the active site.
The importance of the carbamylated lysine for the urease active site building and integrity was investigated through the structural determination of the KAU mutants Lysa217Glu (PDB code 1A5K, Table 1), Lysa217Ala (PDB code 1A5M, Table 1), and Lysa217Cys/Cysa319Ala (PDB code 1A5L, Table 1) . The structures of these mutants reveal the complete absence of bound Ni ions, with the Glu, Ala, and Cys side chains substituting the carbamylated lysine not being able to support the formation of the bimetallic active site. However, all three mutants could be 'chemically rescued' by adding small organic acids, such as formic acid, and Ni ions, a procedure that yielded the active site containing both nickel ions. The structures of the Lysa217Ala-formate-Ni complex (PDB code 1A5N, Table 1, Figure 11) and Lysa217Cys/Cysa319Ala-formate-Ni complex (PDB code 1A5O, Table 1) reveal the presence of a dinuclear Ni center bridged by formate instead of the carbamate group of Lysa217 as in wild type KAU . Only one water molecule (W502) was refined in a position close to Ni(2).
The mutation of Hisa134, one of the two histidine ligands to Ni(2), to the corresponding alanine residue yields the catalytically inactive mutant Hisa134Ala . The structure of the mutant reveals an enzyme lacking Ni(2) and featuring only Ni(1) in the active site (PDB code 1FWI, Table 1, Figure 11) . The Lysa217* is still carbamylated, and the position of all active site residues (except for the missing Hisa134) highlights again the substantial rigidity of the structure. The Ni(1) ion is octahedrally bound to the same protein ligands as in wild-type urease, in addition to three water molecules.
Two residues not directly bound to the nickel ions in KAU, but well integrated in the active site and interacting with the Ni-bound water molecules through H-bonds are Hisa219 and Hisa320. Hisa219 has a direct or indirect role for Hisa219 in substrate binding, as indicated by the much lower affinity for the substrate urea (Km increases ~103-fold) for the Hisa219Ala mutant . On the other hand, Hisa219 is not involved in acid/base catalysis, as indicated by the similar rate of enzymatic urea hydrolysis of the same mutant as well as Hisa219Asn and Hisa219Gln [30, 40]. The structure of this mutant (PDB code 1KRB, Table 1, Figure 11) shows substantial identity with native KAU, including the position of the two water molecules in the vicinity of the Ni ions, except with the obvious absence of Hisa219 and the presence, in its place, of Alaa219 . The importance of Hisa219 in the placement of the water molecule bound to Ni(1), is revealed by the absence of this ligand in the structure of the mutant. On the other hand, for the Hisa219Asn and the Hisa219Gln mutants the decrease in affinity is ~10-fold less marked. This could be due, in principle, to the fact that the latter two mutants retain H-bonding capability, and indeed this is observed in their crystal structures (PDB codes 1EJS and 1EJT for the Hisa219Asn and Hisa219Gln mutant, respectively, Table 1) . Figure 11 shows how the mutated Glna219 residue in 1EJT donates an H-bond through its -NH2 group to the water molecule coordinated to Ni(1), contributing to the building of the tetrahedral cluster of solvent molecules found in the native enzyme (compare with 1FWJ in Figure 4).
The Hisa320Ala, Hisa320Asn, and the Hisa320Gln mutants display only a small change in Km, but a ~104/105-fold decrease in kcat with respect to the wild-type enzyme [30,40]. Furthermore, their activity-pH profiles do not show the Hisa320 dependent pKa = 6.5 observed for wild-type urease [39,40], but rather a shift of the pH optimum to ~6 . The structures of the Hisa320Ala, Hisa320Asn, and Hisa320Glu mutants (PDB codes 1KRC, 1EJU, and 1EJV, respectively, Table 1) have been determined [25,30]. In the case of 1KRC the Ni-bridging hydroxide is still found in place, while the major difference with the wild-type active site consists in the loss of the solvent molecules terminally bound to the nickel ions. This could be ascribed to the removal, in the mutant, of a residue capable to provide support for these molecules through the H-bonding network found in the wild-type enzyme, of which Hisa320 is a key player. In the case of 1EJU and 1EJV, the substitution of Hisa320 with Asn or Gln residues guarantees similar H-bonding properties compared with the native residue. Consistently, the active site structures reveal the presence of the four solvent-derived molecules in locations similar to those found in the wild-type (Figure 11, 1EJU). However, the active site flap becomes disordered and is not visible in the electron density, thereby hindering the localization of the Asna320 or Glna320 residues. In these mutants the capability of acid/base catalysis provided by the imidazole ring of Hisa320 in the native protein is compromised by the presence of the amide functional groups of the mutated residues, thereby justifying the drastic decrease of enzymatic activity.
In the structure of KAU, the carboxylic group of Aspa221 is at H-bonding distance (3.0 A) from the Hisa320 NS atom. This suggests that the latter is protonated, and therefore that the corresponding Hisa320 Ne atom is deprotonated at the crystallization pH of 7.5, very close to the optimum pH for catalytic activity (pH = 8). This could induce a role of proton acceptor for the Hisa320 residue during the catalytic cycle. Mutation of Aspa221 into alanine provokes a 103 decrease of the catalytic rate and a shift of the pH optimum to ~5 . The structure of the Aspa221Ala mutant (PDB code 1EJR, Table 1) reveals a large disorder of the active site flap that contains Hisa320, while all the rest of the active site residues are essentially unchanged. This observation supports a role of Aspa221 in regulating the enzymatic activity through the modulation of the structural and reactivity features of Hisa320.
Cysa319 is located on the flexible flap covering the active site of KAU. This residue is largely conserved in all urease enzymes, except for the enzyme from Staphylococcus xylosus, which has a threonine in this position . Chemical modification of Cysa319 blocks enzyme activity [55,56], indicating that this residue is somehow involved in catalysis. However, the Cysa319Ala mutant is still ~50 % as active as the wild-type urease . Structures of this mutant were determined at pH 6.5, 7.5, 8.5, and 9.4 (PDB codes 1FWB, 1FWA, 1FWC, and 1FWD in that order, Table 1) . There were no significant differences among all these structures. The most evident differences between the structure of Cysa319Ala mutant and that of the wild-type enzyme involve a much-reduced mobility of the flexible flap covering the active site, still found in a 'closed' conformation, but displaying significantly reduced mobility. In contrast, the structures of Cysa319Asp (PDB code 1FWF), Cysa319Ser (PDB code 1FWG), and Cysa319Tyr (PDB code 1FWH) (see Table 1), which display, 0.03, 4.5 and 0%, respectively, of the activity observed for the wild-type, indicate a much higher mobility of the flap, but the same active site environment . In the case of Cysa319Tyr, the flap is in the open conformation as for native BPU, an observation that was related to the presence of the bulky tyrosine side chain .
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