Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe

Kinghorn, James R.; Fluri, R.

doi:10.1007/bf00420225

Cited by 17 publications

(19 citation statements)

References 17 publications

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“…[7][8][9], and an essential role for auxiliary genes in forming active urease has been confirmed in these and many other bacteria. Genetic studies are consistent with a similar requirement for urease accessory genes in the fungi Neurospora crassa (10), Schizosaccharomyces pombe (11), and Aspergillus nidulans (12,13). Finally, two loci (Eu2 and Eu3) that appear to encode genes associated with urease maturation factors such as nickel emplacement have been identified in soybean plants (14).…”

supporting

confidence: 63%

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

Park

Carr

Hausinger

1994

Proc. Natl. Acad. Sci. U.S.A.

111

116

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The formation of active urease in Kiebsiella aerogenes requires the presence ofthree structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickelcontaining enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid contining the K. aerogenes urease gene cluster or derivatives ofthis plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease.

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supporting

confidence: 63%

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

Park

Carr

Hausinger

1994

Proc. Natl. Acad. Sci. U.S.A.

111

116

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“…The addition of 0.1 mM Ni restored the ability of this mutant fungus to grow on urea and increased the urease activity to 5 to 8% of the wildtype levels, but Ni addition to cell extracts did not result in urease activity. Furthermore, the number of urease loci in Neurospora crassa (4) and Schizosaccharomyces pombe (11) exceeds that required for the urease structural genes. The function of the additional genes is unknown, but one could speculate that they function in Ni incorporation or other maturation event.…”

Section: Resultsmentioning

confidence: 99%

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme

1990

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Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79°C) compared with apoenzyme (Tm = 62°C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free ceil cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.Urease, a nickel-containing enzyme found in many plants and microorganisms, hydrolyzes urea to yield carbonic acid and ammonia (2,18). Whereas the role of urease in plants is poorly understood (28), the microbial enzyme plays important roles in human and animal pathogenic states, in ruminant metabolism, and in environmental transformations of certain nitrogenous compounds (18). The best-characterized plant urease is that isolated from jack bean; it was the first enzyme ever crystallized (23) and the first enzyme shown to possess nickel (6). This hexameric protein contains two Ni ions per subunit (Mr = 90,770), the sequence of which was recently reported (24). The best-characterized bacterial urease is that from Klebsiella aerogenes (currently K. pneumoniae), which possesses three subunits [Mrs = 72,000 [a], 11,000 [1], and 9,000 [ry]) in an a2f34Y4 stoichiometry (25).The native enzyme contains two catalytic sites, each of which is associated with two Ni ions (26). The genes for K. aerogenes urease were recently cloned and overexpressed such that urease accounted for over 10% of the cellular protein (20). Although the enzyme requires Ni for activity, no Ni-dependent regulation of gene expression was observed.This report describes the isolation, characterization, and in vivo reconstitution of apourease from K. aerogenes. Experiments were designed to ascertain (i) whether Ni is required for intersubunit association, (ii) whether metal ions other than Ni are incorporated into urease during growth in Ni-free medium, (iii) how the presence of Ni affects the enzyme thermal stability and histidine reactivity, and (iv) the requirements for Ni to be incorporated into preformed apoenzyme. MATERIALS AND METHODSBacterial strains and growth conditions. K. aerogenes CG253 was transformed with plasmid pKAU19 (20), which possesses the urease genes from this microorgani...

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“…The four urease-negative mutants of S. pombe (ure1, ure2, ure3 and ure4) were a gift from Dr. R. Fluri (Kinghorn and Fluri 1984). All were leucine auxotrophs (leu1-32).…”

Section: Biological Materialsmentioning

confidence: 99%

“…Complementation of S. pombe urease mutants by genomic DNA bearing the putative accessory genes ureD, ureF and ureG Three non-allelic urease-negative mutants -ure1, ure3, and ure4 (Kinghorn and Fluri 1984;Lubbers et al 1997) -were transformed with a genomic DNA library constructed in the plasmid pDB248' (Beach et al 1982) and clones expressing the plasmid marker (LEU2) were selected on EMM minimal medium without leucine supplementation. Clones were then transferred to EMM lacking leucine, in which the nitrogen source was 5 mM urea (EMMU).…”

Section: Identification Of Putative Accessory Genes In S Pombementioning

confidence: 99%

“…S. pombe produces a ''plant-like'' urease with an $90-kDa subunit (Tange and Niwa 1997), and at least four, non-allelic, urease-negative mutants have been identified (Kinghorn andFluri 1984, Lubbers et al 1997). We identified candidate genes encoding S. pombe UreD, UreF and UreG and tested the functional activity of soybean UreD and UreF in the corresponding S. pombe ureasedeficient mutants.…”

Section: Introductionmentioning

confidence: 99%

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Activation of the urease of SCHIZOSACCHAROMYCES POMBE by the UreF accessory protein from soybean

Bacanamwo

Cp²,

Lubbers

et al. 2002

Mol Gen Genomics

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Plant orthologs of the bacterial urease accessory genes ureD and ureF, which are required for the insertion of the nickel ion at the active site, have been isolated from soybean ( Glycine max L. Merr.), tomato ( Lycopersicon esculentum) and Arabidopsis thaliana. The functionality of soybean UreD and UreF was tested by measuring their ability to complement urease-negative mutants of Schizosaccharomyces pombe, a eukaryote which produces a "plant-like" urease of ~90 kDa. The S. pombe ure4 mutant was complemented by a 12-kb fragment of S. pombe genomic DNA, which was shown by PCR to contain a putative ureD gene. However, ure4 was not complemented by a UreD cDNA soybean, expressed under the control of a strong promoter. In contrast, an S. pombe ure3 mutation was complemented by both a 10-kb fragment of S. pombe DNA containing ureF and the UreF cDNA from soybean. Soybean Eu2 is a candidate urease accessory gene; its product cooperates with the Eu3 protein in activating apourease in vitro. However, the sequences of UreD and UreF transcripts from two eu2/eu2 mutants, recovered as RT-PCR products, revealed no mutational alteration, suggesting that Eu2 encodes neither UreD nor UreF.

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Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe

Cited by 17 publications

References 17 publications

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme

Activation of the urease of SCHIZOSACCHAROMYCES POMBE by the UreF accessory protein from soybean

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