Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.
Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.
Mulrooney
S B
SB
Department of Biochemistry, Michigan State University, East Lansing 48824.
Lynch
M J
MJ
Mobley
H L
HL
Hausinger
R P
RP
D001345:Autoradiography; D002853:Chromatography, Liquid; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008970:Molecular Weight; D011511:Proteus / Q000201:enzymology*; D011532:Providencia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014510:Urease / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D014510:Urease