Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis

Abstract

The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.

In Vivo and in Vitro Kinetics of Metal Transfer by the Klebsiella aerogenes Urease Nickel Metallochaperone, UreE

“…The lag period observed in the deletion strain highlights the importance of UreE for the rapid transfer of Ni 2ϩ into preformed urease apoprotein. On the other hand, these studies reinforce previous conclusions that UreE is not required for urease activation (18,23). With the exception of cells containing H96A/H144*UreE, all cultures possessing H144*UreE variants demonstrated intermediate abilities to activate urease with greatest inhibition being noted for cells producing the H110A, D111A, and H112A variants.…”

“…Also in bacteria, three distinct Ni 2ϩ metallochaperones have been tentatively identified (12). HypB, essential for Ni 2ϩ insertion into hydrogenase by a process that requires GTP hydrolysis (13,14), binds 18 Ni 2ϩ /dimer (K d ϭ 2.3 M) as isolated from Bradyrhizobium japonicum (15). Similarly, Rhodospirillum rubrum CooJ, which assists with CO dehydrogenase activation (16), binds 4 Ni 2ϩ /monomer (K d ϭ 4.3 M) (17).…”

“…Similarly, Rhodospirillum rubrum CooJ, which assists with CO dehydrogenase activation (16), binds 4 Ni 2ϩ /monomer (K d ϭ 4.3 M) (17). Finally, of particular interest to the work described here is Klebsiella aerogenes UreE, the putative urease metallochaperone (18) that binds 5-6 Ni 2ϩ /dimer (average K d ϭ ϳ10 M) (19). In each case, mutations within the corresponding genes are phenotypically suppressed by increasing the Ni 2ϩ concentration in the medium (16,18,20).…”

“…Finally, of particular interest to the work described here is Klebsiella aerogenes UreE, the putative urease metallochaperone (18) that binds 5-6 Ni 2ϩ /dimer (average K d ϭ ϳ10 M) (19). In each case, mutations within the corresponding genes are phenotypically suppressed by increasing the Ni 2ϩ concentration in the medium (16,18,20). Although all three proteins possess histidine-rich regions, they exhibit no overall sequence similarity.…”

Visualization of a radical B 12 enzyme with its G-protein chaperone

“…Mutations in MMAA and MCM result in methylmalonic aciduria, a genetically inherited metabolic disease that is devastating in newborns and infants (7). Bacterial homologs of MMAA are involved in assembly of other metalloproteins and include HypB for hydrogenase (8) and UreG for urease (9). These G-protein chaperones bind to their target proteins and then use their GTP hydrolase (GTPase) activity to assist maturation of the target with high specificity (10)(11)(12).…”