Structures of the Klebsiella aerogenes Urease Apoenzyme and Two Active-Site Mutants,

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Abstract
Urease from Klebsiella aerogenes [Jabri et al. (1995) Science 268, 998−1004] is an (αβγ)3 trimer with each α-subunit having an (αβ)8-barrel domain containing a binickel active center. Here we examine structure−function relations for urease in more detail through structural analysis of the urease apoenzyme at 2.3 Å resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 Å resolution. With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel. In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Aspα221 could contribute to the lower activity of H219A. In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position. This shift shows that the nickel ligation is rather sensitive to the environment and the change in ligation may contribute to the 105-fold lower activity of H320A. In addition, these results show that urease is resilient to the loss of nickel ions and mutations. Analysis of the urease tertiary/quaternary structure suggests that the stability of this enzyme may be largely due to its burial of an unusually large fraction of its residues: 50% in the γ-subunit, 30% in the β-subunit, and 60% in the α-subunit.