Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria

Sakamoto, Hiroshi; Landais, Stéphanie; Evrin, Cécile; Laurent‐Winter, Christine; Bârzu, Octavian; Kelln, Rod A.

doi:10.1099/mic.0.26996-0

Cited by 14 publications

(10 citation statements)

References 26 publications

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“…Dependence of UMP Kinase Activity on ATP Concentration-E. coli and S. typhimurium UMP kinases were shown to exhibit hyperbolic dependence of activity as a function of ATP concentration in both the absence and presence of GTP (1,20). The same was true for N. meningitidis UMP kinase.…”

Section: Resultsmentioning

confidence: 82%

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

Evrin

Străuţ

Slavova‐Azmanova

et al. 2007

Journal of Biological Chemistry

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In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K m for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Grampositive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T % R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.Bacterial UMP kinases represent a particular subfamily of NMP 2 kinases (1, 2). They do not share any significant sequence homology with other known NMP kinases and exist in solution as stable hexamers. A first structural model of Escherichia coli UMP kinase (3) based on the conservation of the carbamate kinase and N-acetylglutamate kinase folds (4, 5) helped to better rationalize previous site-directed mutagenesis experiments (6). The crystal structure of E. coli UMP kinase (7) indicated a similar fold between its monomers and N-acetylglutamate kinase, a dimeric enzyme (4, 5). However, the quaternary structure assembly of these two proteins is completely different (7). Deposited crystal structures of UMP kinases from other bacteria such as Pyrococcus furiosus (8), Neisseria meningitidis (Protein Data Bank code 1YBD), Hemophilus influenzae (code 2AIF), and Streptococcus pyogenes (code 1Z9D) show threedimensional structures very similar to that of the E. coli enzyme. The residues essential for binding nucleotide substrates and catalysis are conserved among all bacterial UMP kinases ( Fig. 1) (3, 9). Consequently, the active sites of these enzymes and the phosphoryl transfer mechanisms are most probably similar.Comparison of the biochemical properties of recombinant UMP kinases from Gram-negative E. coli (1, 2) and Gram-positive Streptococcus pneumoniae (10) indicated significant differences in their kinetic properties particularly in their regulation by nucleotides. Unlike the E. coli enzyme, U...

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Section: Resultsmentioning

confidence: 82%

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

Evrin

Străuţ

Slavova‐Azmanova

et al. 2007

Journal of Biological Chemistry

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“…30 The allosteric pocket of the B. anthracis enzyme, with its atypical configuration of side chains, may provide a particularly suitable site for pharmacological intervention.…”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure of UMP Kinase from Bacillus anthracis (BA1797) Reveals an Allosteric Nucleotide-Binding Site

Meier

Carter

Sainsbury

et al. 2008

Journal of Molecular Biology

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“…Sequence comparisons indicate that this enzyme belongs to the amino acid kinase protein family (AAKF) 9 (PF00696 †), a family in which the two structurally characterized members, carbamate kinase (CK) and N-acetyl-L-glutamate kinase (NAGK), [9][10][11] have a characteristic fold and a homodimeric architecture, and catalyse acyl group phosphorylation. However, UMPK phosphorylates a phosphate group, it is hexameric 4,8,12 and is the subject of feed-back regulation, 4,8,12,13 mediated by UTP (inhibitor) and GTP (activator). These characteristics render prokaryotic UMPK particularly interesting for structural study, specially since other enzymes of the AAKF, including aspartokinase, glutamate-5-kinase, 14,15 N-acetyl-L-glutamate synthase, 16 and even the NAGK of most organisms (but not that of Escherichia coli, the NAGK for which the structure was reported 17 ) are the targets of feed-back regulation, and since some of these enzymes are hexameric.…”

Section: Introductionmentioning

confidence: 99%

The Crystal Structure of Pyrococcus furiosus UMP Kinase Provides Insight into Catalysis and Regulation in Microbial Pyrimidine Nucleotide Biosynthesis

Marco-Marı́n

Gil-Ortiz

Rubio

2005

Journal of Molecular Biology

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Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria

Cited by 14 publications

References 26 publications

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

The Crystal Structure of UMP Kinase from Bacillus anthracis (BA1797) Reveals an Allosteric Nucleotide-Binding Site

The Crystal Structure of Pyrococcus furiosus UMP Kinase Provides Insight into Catalysis and Regulation in Microbial Pyrimidine Nucleotide Biosynthesis

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