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Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

ABSTRACT: A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. Six closely spaced open reading frames were found: ureA (encoding a peptide of 11.1 kilodaltons [kDa]), ureB (11.7-kDa peptide), ureC (60.3-kDa peptide), ureE (17.6-kDa peptide), ureF (25.2-kDa peptide), and ureG (21.9-kDa peptide). Immediately after the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are encoded by ureA, ureB, and ureC based on protein structural studies and sequence homology to jack bean urease. Potential roles for ureE, ureF, and ureG were explored by deleting these accessory genes from the operon. The deletion mutant produced inactive urease, which was partially purified and found to have the same subunit stoichiometry and native size as the active enzyme but which contained no significant levels of nickel. The three accessory genes were able to activate apo-urease in vivo when they were cloned into a compatible expression vector and cotransformed into cells carrying the plasmid containing ureA, ureB, and ureC. Thus, one or more of the ureE, ureF, or ureG gene products are involved in nickel incorporation into urease.

SUBMITTER: Mulrooney SB

PROVIDER: S-EPMC526901 | biostudies-other | 1990 Oct

REPOSITORIES: biostudies-other

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Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

Mulrooney S B SB Hausinger R P RP

Journal of bacteriology 19901001 10

A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. Six closely spaced open reading frames were found: ureA (encoding a peptide of 11.1 kilodaltons [kDa]), ureB (11.7-kDa peptide), ureC (60.3-kDa peptide), ureE (17.6-kDa peptide), ureF (25.2-kDa peptide), and ureG (21.9-kDa peptide). Immediately after the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are enco ...[more]

PMID: 2211515

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Project description:Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreB?1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreB?1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.

Dataset Information

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

Publications

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

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