Mechanism for Attenuation of DNA Binding by MarR Family Transcriptional Regulators by Small Molecule Ligands

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.     

The crystal structure of the transcriptional regulator HucR from Deinococcus radiodurans reveals a repressor preconfigured for DNA binding

We report here the 2.3 A resolution structure of the hypothetical uricase regulator (HucR) from Deinococcus radiodurans R1. HucR, a member of the MarR family of DNA-binding proteins, was previously shown to repress its own expression as well as that of a uricase, a repression that is alleviated both in vivo and in vitro upon binding uric acid, the substrate for uricase. As uric acid is a potent scavenger of reactive oxygen species, and as D. radiodurans is known for its remarkable resistance to DNA-damaging agents, these observations indicate a novel oxidative stress response mechanism. The crystal structure of HucR in the absence of ligand or DNA reveals a dimer in which the DNA recognition helices are preconfigured for DNA binding. This configuration of DNA-binding domains is achieved through an apparently stable dimer interface that, in contrast to what is observed in other MarR homologs for which structures have been determined, shows little conformational heterogeneity in the absence of ligand. An additional amino-terminal segment, absent from other MarR homologs, appears to brace the principal helix of the dimerization interface. However, although HucR is preconfigured for DNA binding, the presence of a stacked pair of symmetry-related histidine residues at a central pivot point in the dimer interface suggests a mechanism for a conformational change to attenuate DNA binding.     

The SarA protein family of Staphylococcus aureus

Staphylococcus aureus is widely appreciated as an opportunistic pathogen, primarily in hospital-related infections. However, recent reports indicate that S. aureus infections can now occur in other wise healthy individuals in the community setting. The success of this organism can be attributed to the large array of regulatory proteins, including the SarA protein family, used to respond to changing microenvironments. Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: (1) single domain proteins; (2) double domain proteins; (3) MarR homologs. Structural studies have also demonstrated that SarA, SarR, SarS, MgrA and thus possibly all members of this protein family are winged helix proteins with minor variations. Mutagenesis studies of SarA disclose that the winged helix motifs are important for DNA binding and function. Recent progress concerning the functions and plausible mechanisms of regulation of SarA and its homologs are discussed.     

Inhibition of xanthine oxidase by pterins

The effect of a panel of pterins on xanthine oxidase was investigated by measuring formation of urate from xanthine as well as formazan production from nitroblue tetrazolium. The pterin derivatives, depending on their chemical structure, decreased urate as well as formazan generation: 200 μM neopterin and biopterin suppressed urate formation (90% from baseline) and formazan production (80% from baseline) as well. Their reduced forms, 7,8-dihydroneopterin and 5,6,7,8-tetrahydrobiopterin, showed a lesser but still strongly diminishing influence (40% from baseline). Another oxidized pterin namely leukopterin showed only a weak inhibitory effect. Xanthopterin, a known substrate of xanthine oxidase, had a strong effect on urate formation (80% inhibition), but a lesser effect on formazan production (30% reduction). When iron-(III)-EDTA complex was added to the reaction mixture all the effects were more pronounced. Superoxide dismutase, which removes superoxide anion by dismutation intooxygen, decreased formazan production in addition to pterin derivatives and had a small but enhancing effect on urate formation. Also the reductant N-acetylcysteine had an additive effect to pterins to diminish formazan production in a dose-dependent way. The results of our study suggest that depending on their chemical structure pterins reduce superoxide anion generation by xanthine oxidase.     

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

ST1710–DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators

ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 Å resolutions, respectively. The ST1710–DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710–DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism.     

Management of Hyperuricemia with Rasburicase Review

Tumor lysis syndrome (TLS) is a serious complication in patients with hematological malignancies. Massive lysis of tumor cells can lead to hyperuricemia, hyperkalemia, hyperphosphatemia and hypocalcaemia. These metabolic disturbances may result in renal failure, because of precipitation of uric acid crystals and calcium phosphate salts in the kidney. The standard prophylaxis or treatment of hyperuricemia consists of decreasing uric acid production with allopurinol and facilitating its excretion by urinary alkalinization and hyperhydration. By inhibiting the enzyme xanthine oxidase, allopurinol blocks the conversion of hypoxanthine and xanthine into uric acid. An alternative treatment is urate oxidase which oxidates uric acid into allantoin. Allantoin is 5–10 times more soluble than uric acid and is therefore excreted easily. In several clinical trials rasburicase, the recombinant form of urate oxidase, has shown to be very effective in preventing and treating hyperuricemia. Rasburicase, in contrast with the non‐recombinant form of urate oxidase uricozyme, is associated with a low incidence of hypersensitivity reactions. In addition to the demonstrated clinical benefit, rasburicase also proved to be a cost‐effective option in the management of hyperuricemia.     

Management of hyperuricemia with rasburicase review

Tumor lysis syndrome (TLS) is a serious complication in patients with hematological malignancies. Massive lysis of tumor cells can lead to hyperuricemia, hyperkalemia, hyperphosphatemia and hypocalcaemia. These metabolic disturbances may result in renal failure, because of precipitation of uric acid crystals and calcium phosphate salts in the kidney. The standard prophylaxis or treatment of hyperuricemia consists of decreasing uric acid production with allopurinol and facilitating its excretion by urinary alkalinization and hyperhydration. By inhibiting the enzyme xanthine oxidase, allopurinol blocks the conversion of hypoxanthine and xanthine into uric acid. An alternative treatment is urate oxidase which oxidates uric acid into allantoin. Allantoin is 5-10 times more soluble than uric acid and is therefore excreted easily. In several clinical trials rasburicase, the recombinant form of urate oxidase, has shown to be very effective in preventing and treating hyperuricemia. Rasburicase, in contrast with the non-recombinant form of urate oxidase uricozyme, is associated with a low incidence of hypersensitivity reactions. In addition to the demonstrated clinical benefit, rasburicase also proved to be a cost-effective option in the management of hyperuricemia.