Genetics of nickel ion incorporation into Proteus mirabilis urease

Abstract

Proteus mirabilis urease, a nickel metalloenzyme, is essential for virulence of this bacterial species in the urinary tract. The urease gene cluster contains eight genes including a regulatory gene (ureR), three enzyme structural genes (ureABC), and four accessory genes (ureDEFG). The urease accessory gene products have been implicated in the process of nickel incorporation. We examined the involvement of two of these accessory genes, ureG and ureE, in expression of an active urease and nickel incorporation. In addition we studied the coordination of nickel ions by the urease structural gene, ureC. First we studied the essential role of ureG in the production of active urease. Escherichia coli carrying cloned urease genes, ureDABCEFG, displays urease activity when cultured in M9 minimal medium. Clones lacking sequences downstream of ureF (i.e., ureG) were urease-negative. Deletion mutations were constructed. Urease activity of the ureG mutant was undetectable. We speculate that UreG may play an energy-dependent chaperonin-like role in the insertion of nickel ions into urease apoenzyme. Next we examined the role of ureE. The accessory protein UreE, appears to act as a nickel binding protein for the urease gene cluster. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by addition of 5 {dollar}\mu{dollar}M NiCI{dollar}\sb2{dollar} to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues suggested that UreE may act as a Ni{dollar}\sp{lcub}2+{rcub}{dollar} chelator for the urease operon. UreE was purified yielding single polypeptide of 20 kDa apparent molecular size. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated to be 36 kDa, suggesting that the protein acts as a dimer. These data suggest that UreE is a Ni{dollar}\sp{lcub}2+{rcub}{dollar}-binding protein that is necessary for synthesis of a catalytically active urease especially at low Ni{dollar}\sp{lcub}2+{rcub}{dollar} concentrations. Finally, we examined the coordination of Ni{dollar}\sp{lcub}2+{rcub}{dollar} by residues in the UreC enzyme structural subunit. An amino acid sequence within the large urease subunit, UreC, is highly conserved for ureases and has been suggested to reside within the enzyme active site. Histidine residues ha8ve been postulated to play a role in catalysis by coordinating Ni{dollar}\sp{lcub}2+{rcub}{dollar} ions. Oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of {dollar}\sp{lcub}63{rcub}{dollar}Ni{dollar}\sp{lcub}2+{rcub}{dollar} incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC, His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni{dollar}\sp{lcub}2+{rcub}{dollar} into apoenzyme. Five other histidine mutants expressed detectable amount of urease activities. However, alteration His-321 and Cys-319 abolished catalytic activity. These two residues may play significant roles in urea hydrolysis as well. (Abstract shortened by UMI.)