Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme.
Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ 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 Ni2+ into apoenzyme.
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
B
B
Sriwanthana
H L
HL
Mobley
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D017124:Conserved Sequence; D006639:Histidine / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism*; D011513:Proteus mirabilis / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D014508:Urea / Q000378:metabolism*; D014510:Urease / Q000235:genetics / Q000378:metabolism*
D006639:Histidine; D009532:Nickel; D014508:Urea; D014510:Urease