Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids

Ho, Ya-Yeh; Huang, Yen‐Hua; Huang, Cheng-Yang

doi:10.1007/s00726-012-1451-3

Cited by 32 publications

(34 citation statements)

References 51 publications

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“…; Ho et al . ) revealed the degree of variability at each position along the primary sequence. Amino acid residues that are highly variable are colored teal, whereas highly conserved amino acid residues are colored burgundy.…”

Section: Resultsmentioning

confidence: 99%

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DnaT is a single‐stranded DNA binding protein

2013

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DnaT is one of the replication restart primosomal proteins required for reinitiating chromosomal DNA replication in bacteria. In this study, we identified and characterized the singlestranded DNA (ssDNA)-binding properties of DnaT using electrophoretic mobility shift analysis (EMSA), bioinformatic tools and two deletion mutant proteins, namely, DnaT26-179 and DnaT42-179. ConSurf analysis indicated that the N-terminal region of DnaT is highly variable. The analysis of purified DnaT and the deletion mutant protein DnaT42-179 by gel filtration chromatography showed a stable trimer in solution, indicating that the N-terminal region, amino acid 1-41, is not crucial for the oligomerization of DnaT. Contrary to PriB, which forms a single complex with a series of ssDNA homopolymers, DnaT, DnaT26-179 and DnaT42-179 form distinct complexes with ssDNA of different lengths and the size of binding site of 26 AE 2 nucleotides (nt). Using bioinformatic programs (PS) 2 and the analysis of the positively charged/hydrophobic residue distribution, as well as the biophysical results in this study, we propose a binding model for the DnaT trimer-ssDNA complex, in which 25-nt-long ssDNA is tethered on the surface groove located in the highly conserved C-terminal domain of DnaT. These results constitute the first study regarding ssDNA-binding activity of DnaT. Consequently, a hand-off mechanism for primosome assembly was modified.

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Section: Resultsmentioning

confidence: 99%

“…The recombinant DnaT proteins were expressed and purified using the protocol described previously for allantoinase (Ho et al . ), hydantoinase (Huang et al . ), dihydroorotase (Wang et al .…”

Section: Methodsmentioning

confidence: 99%

DnaT is a single‐stranded DNA binding protein

2013

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“…However, the existence of fully functional DHOases with a mononuclear metal center is supported by the fact that deletion of the β-site in A aeolicus DHOase [8] by replacing the two histidines binding the β Zn with Ala affected neither catalysis nor active site structure. However, when the second metal binding site in amidohydrolases with a binuclear metal center was eliminated, all catalytic activity ceased [31, 32]. Nonetheless, we have unambigiously shown that M. jannaschii DHOase contains two Zn ions.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Dihydroorotase from Methanococcus jannaschii

2017

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The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC-MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with Vmax = 12.2 µmol min−1 mg−1 and Km = 0.14 mM at 25° C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85° C and 90° C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85° C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability – more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined.

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“…In contrast, the replacement of the corresponding histidines in the β site of Klebsiella pneumoniae dihydroorotase [18] and allantoinase [19], both of which have a binuclear metal site, completely abolished catalytic activity. The crystal structure of the active double mutant showed a zinc atom in the α-site and a water molecule Jβ (HOH2080/A) near the β-site (Figure 2B, Figure 6) - thus eliminating the unlikely possibility that the one zinc atom was randomly distributed between the two sites.…”

Section: Discussionmentioning

confidence: 99%

“…The proteins were expressed individually in BL21 (DE3) and purified by Ni +2 affinity chromatography on a 1-ml Ni +2 Probond column (Invitrogen). While the DHO from some organisms has been found to have limited thermal stability [4,19], the DHO and ATC from A. aeolicus , a hyperthermophile that grows at 95°C [34], are stable for several months when stored in 50 mM TrisHCl, 200 mM NaCl, 200–300 mM imidazole, pH 8.0 at 4°C.…”

Section: Methodsmentioning

confidence: 99%

The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

et al. 2013

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BackgroundDihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion.ResultsZinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the β-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the β-site was eliminated but that the active site remained otherwise intact.ConclusionsThus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.

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Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids

Cited by 32 publications

References 51 publications

DnaT is a single‐stranded DNA binding protein

DnaT is a single‐stranded DNA binding protein

Characterization of the Dihydroorotase from Methanococcus jannaschii

The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

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