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D-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations.

ABSTRACT: The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K⁺, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K⁺ and Rb⁺ ions. A strict dependence on ionic radius of the MVC was observed, with Li⁺ and Na⁺ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K⁺, Rb⁺, or Cs⁺ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

SUBMITTER: Pederick JL

PROVIDER: S-EPMC7278361 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The ATP-grasp enzymes.

Project description:The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+β domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis, and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism, and inhibition of the ATP-grasp enzymes.

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Molecular Mechanisms of Enzyme Activation by Monovalent Cations.

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6-Arylpyrido[2,3-d]pyrimidines as novel ATP-competitive inhibitors of bacterial D-alanine:D-alanine ligase.

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| S-EPMC3410885 | biostudies-literature

Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens.

Project description:Using degenerate oligonucleotides complementary to sequences encoding conserved amino acid motifs in D-alanine-D-alanine (Ddl) ligases, we have amplified ca. 600-bp fragments from Enterococcus casseliflavus ATCC 25788 and Enterococcus flavescens CCM439. Sequence analysis of the amplification products indicated that each strain possessed two genes, ddlE. cass. and vanC-2, and ddlE. flav. and vanC-3, respectively, encoding Ddl-related enzymes. The fragments internal to the vanC genes were 98.3% identical. The vanC-2 gene was cloned into Escherichia coli and sequenced. Extensive similarity (66% nucleotide identity) was detected between this gene and vanC-1 from Enterococcus gallinarum (S. Dutka-Malen, C. Molinas, M. Arthur, and P. Courvalin, Gene 112:53-58, 1992), suggesting that the vanC genes are required for intrinsic low-level resistance to vancomycin. The partial deduced amino acid sequences of ddlE. cass. and ddlE. flav. were identical and closely related to that of the Ddl ligase of Enterococcus faecalis (79% identity). In Southern hybridization experiments, only DNA from E. casseliflavus and E. flavescens hybridized to probes internal to the vanC-2 and ddlE. cass. genes.

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Competitive interaction of monovalent cations with DNA from 3D-RISM.

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ADP-Mg2+ bound to the ATP-grasp domain of ATP-citrate lyase.

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| S-EPMC3212355 | biostudies-literature

Measurement of the effect of monovalent cations on RNA hairpin stability.

Project description:Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A.U and G.C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (<15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G.C helices were unaffected by changing salt concentration, A.U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U.C.U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.

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Acinetobacter baumannii increases tolerance to antibiotics in response to monovalent cations.

Project description:Acinetobacter baumannii is well adapted to the hospital environment, where infections caused by this organism are associated with significant morbidity and mortality. Genetic determinants of antimicrobial resistance have been described extensively, yet the mechanisms by which A. baumannii regulates antibiotic resistance have not been defined. We sought to identify signals encountered within the hospital setting or human host that alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as being an important signal that induces significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin upon the culturing of A. baumannii cells in physiological NaCl concentrations. Proteomic analyses of A. baumannii culture supernatants revealed the release of outer membrane proteins in high NaCl, including two porins (CarO and a 33- to 36-kDa protein) whose loss or inactivation is associated with antibiotic resistance. To determine if NaCl affected expression at the transcriptional level, the transcriptional response to NaCl was determined by microarray analyses. These analyses highlighted 18 genes encoding putative efflux transporters that are significantly upregulated in response to NaCl. Consistent with this, the effect of NaCl on the tolerance to levofloxacin and amikacin was significantly reduced upon the treatment of A. baumannii with an efflux pump inhibitor. The effect of physiological concentrations of NaCl on colistin resistance was conserved in a panel of multidrug-resistant isolates of A. baumannii, underscoring the clinical significance of these observations. Taken together, these data demonstrate that A. baumannii sets in motion a global regulatory cascade in response to physiological NaCl concentrations, resulting in broad-spectrum tolerance to antibiotics.

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A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.

Project description:Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4). a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core. Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products.

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| S-EPMC4388272 | biostudies-literature

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