Purification and characterization of <i>Klebsiella aerogenes</i> UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly

Lee, Mann Hyung; Pankratz, H. Stuart; Wang, Shengke; Scott, Robert A.; Finnegan, Michael G.; Johnson, Michael K.; Ippolito, Joseph A.; Christianson, D.W.; Hausinger, Robert P.

doi:10.1002/pro.5560020617

Cited by 151 publications

(145 citation statements)

References 41 publications

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“…The quadridentate chelating matrix NTA-agarose is the basis of this method. When charged with Ni2+ (coordination number of six), this matrix showed strong specificity for peptides and proteins with contiguous histidines (16 (19). Extended X-ray absorption fine-structure and variable-temperature magnetic circular dichroism analyses of Ni-saturated UreE protein are compatible with a coordination sphere of three to five imidazole rings for each UreE-bound nickel ion (19) (27).…”

Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exmentioning

confidence: 94%

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

et al. 1994

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The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argiieso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 + 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency.Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. eguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. kguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.Rhizobium leguminosarum bv. viciae possesses an H2 uptake system that is able to oxidize H2 generated by the nitrogenase complex as a byproduct of the N2 reduction reaction (8,40). This system consists of an uptake [NiFe] hydrogenase and accessory proteins, and it is only expressed in the plant symbiotic state. The main features of the system have been studied by our laboratory in the Pisum-pea bacteroid symbiosis. The genetic determinants for the H2 uptake system are clustered in a 15-kb DNA region (hup region) in the symbiotic plasmid (21,22). This region has been sequenced, and 17 potential genes have been identified. The first six genes constitute the hydrogenase structural operon and include the genes hupS and hupL, encoding the hydrogenase polypeptides (13), and four additional genes, hupCDEF (14). A five-gene cluster containing hupGHIJK has been identified downstream the hydrogenase structural operon (38 Purification of the HypB protein by Ni(II)-NTA-agarose chromatography. The Ni(II)-nitrilotriacetic acid (NTA)-agarose matrix was obtained from Diagen (Dusseldorf, Germany), and the manufacturer's recommendations for its use (12) were followed, with minor modifications as follows.(i) Denaturing conditions. Frozen cells from a 100-ml induced culture were lysed in the presence of 6 M guanidineHCl (3.5 ml), and cell extracts were applied to an Ni(II)-NTAagarose column (2 by 0.8 cm). Proteins were stepwise eluted by means of buffers of decreasing pH, 8.0, 6.3, 5.9, and 4.5, all of which contained 8 M urea, at a flow rate of 0.5 ml min-1. Fractions (1 ml) were collected, and portions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (18).(ii) Nondenaturing conditions. All the manipulations were carri...

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Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exmentioning

confidence: 94%

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

et al. 1994

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“…KaUreG and KaUreF form a super-complex with the K. aerogenes UreD-apo-urease aggregate (10), suggesting that such large complexes could be required for in vivo activation of urease (9). Finally, KaUreE, the only urease accessory protein shown to bind Ni 2ϩ ions (11)(12)(13)(14), is thought to interact with the K. aerogenes apo-urease-UreD-UreF-UreG super-complex and facilitate Ni 2ϩ incorporation in the urease active site (5). KaUreE possesses a His-rich C terminus (10 of the last 15 residues are histidines) and is able to bind approximately six Ni 2ϩ per dimer in a coordination environment of three to five histidines, with an average K d of ϳ10 M (11).…”

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confidence: 99%

Structural Basis for Ni2+Transport and Assembly of the Urease Active Site by the Metallochaperone UreE from Bacillus pasteurii

Remaut¹,

Safarov²,

Ciurli³

et al. 2001

Journal of Biological Chemistry

105

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Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni 2؉ ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 Å resolution, using SIRAS phases derived from a Hg 2؉ -derivative. BpUreE is composed of distinct N-and Cterminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn 2؉ ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn 2؉ ion can be replaced by Ni 2؉ as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl 2 . A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.The molecular details of the mechanism by which specific metal co-factors are transported in the cell and sorted into the correct metalloenzyme, among the many biological systems that employ transition metal ions for their function without being involved in alternative adventitious and possibly poisonous binding, are still rather unclear. The role of a new class of soluble metal-binding proteins, known as "metallochaperones

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“…Urease within the UreDFG-apourease complex can be fully activated in the presence of nickel, bicarbonate (for lysine carbamylation), GTP, and UreE (13). The latter protein is proposed to function as a "metallochaperone" by delivering nickel ions to UreDFG-apourease (14).…”

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confidence: 99%

“…The wild-type protein binds approximately six nickel ions per homodimer in distorted octahedral geometry with an average of three-five histidine donors per metal ion (14,16). Most of these ligands are presumed to derive from the C-terminal fragment that contains 10 histidines within the last 15 residues.…”

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confidence: 99%

Crystal Structure of Klebsiella aerogenesUreE, a Nickel-binding Metallochaperone for Urease Activation

Song

Mulrooney

Huber

et al. 2001

Journal of Biological Chemistry

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UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multiwavelength anomalous diffraction and refined it to 1.5 Å resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.Urease (EC 3.5.1.5) is a nickel-containing enzyme that catalyzes the hydrolysis of urea to produce ammonia and carbamate (1). Increased pH arising from this reaction is critical to the virulence of several human and animal pathogens (2). The crystal structure of urease from Klebsiella aerogenes provided the first three-dimensional model of the protein and revealed a unique dinuclear active site with the metal ions bridged by a carbamylated lysine residue (3, 4). Subsequent investigations of ureases from Bacillus pasteurii (5) and Helicobacter pylori (6) show essentially identical active site structures. Proper assembly of this metallocenter is a key step for maturation of urease and, in K. aerogenes, involves the products of the ureD,

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Purification and characterization of Klebsiella aerogenes UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly

Cited by 151 publications

References 41 publications

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

Structural Basis for Ni2+Transport and Assembly of the Urease Active Site by the Metallochaperone UreE from Bacillus pasteurii

Crystal Structure of Klebsiella aerogenesUreE, a Nickel-binding Metallochaperone for Urease Activation

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