Crystal Structures of Trypanosoma brucei and Staphylococcus aureus Mevalonate Diphosphate Decarboxylase Inform on the Determinants of Specificity and Reactivity

Byres, E.; Alphey, M.S.; Smith, Terry; Hunter, William N.

doi:10.1016/j.jmb.2007.05.094

Cited by 34 publications

(55 citation statements)

References 55 publications

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“…S. solfataricus is the only example of archaea that has been proven to date to possess the classical MVA pathway (22). The crystal structures of DMD have been solved with the enzymes from several eukaryotes (Saccharomyces cerevisiae [27], Trypanosoma brucei [28], Homo sapiens [29], and Mus musculus), and bacteria (Staphylococcus aureus [28], Staphylococcus epidermidis [30,31], Streptococcus pyogenes, and Legionella pneumophila). Most of the known DMD structures have been reported as homodimers, as elucidated biochemically for some eukaryotic enzymes (32)(33)(34), while DMD from Trypanosoma brucei was shown to be monomeric (28).…”

Section: Importancementioning

confidence: 99%

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2015

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In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ␤-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCEThis study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

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Section: Importancementioning

confidence: 99%

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2015

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“…1 and Table 1). Table 1 also includes kinetic parameter data for previously uncharacterized S. aureus and T. brucei MDD proteins (for which unliganded structures have been determined (13)), demonstrating that these proteins efficiently catalyze the MDD reaction.…”

Section: Kinetic Characterization and Crystal Structure Of Mdd Frommentioning

confidence: 99%

“…In two cases in particular (i.e. S. aureus and T. brucei (13)), attempts to obtain an informative ligand-bound structure were unsuccessful, presumably because of the presence of well ordered sulfate ions found within the purported active site cleft of these enzymes (13). Examination of additional MDD structures available in the PDB suggests that moderate to high concentrations of sulfate anions indeed promote crystallization of these enzymes, as five of the seven structures resulted from crystals harvested from sulfate-containing mother liquids.…”

Section: Kinetic Characterization and Crystal Structure Of Mdd Frommentioning

confidence: 99%

“…These enzymes are characterized by a cone-shaped fold, where the ␤-grasp-like N-terminal region packs orthogonally against the relatively planar C-terminal region comprised of five prominent ␣-helices (13). High resolution crystal structures of MDD proteins have been reported from a number of species across several kingdoms of life, including S. aureus (13), Trypanosoma brucei (13), Saccharomyces cerevisiae (12), as well as Homo sapiens (11). These structures reveal the presence of a deep cleft that forms between the roughly equal halves of the enzyme, which houses the catalytic Asp and Arg residues.…”

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Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Barta

Skaff

McWhorter

et al. 2011

Journal of Biological Chemistry

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The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Grampositive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Å resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K i values observed for these inhibitors. Inspection of enzyme/ inhibitor structures identified the side chain of invariant Ser 192 as making potential contributions to catalysis. Significantly, Ser 3 Ala substitution of this side chain decreases k cat by ϳ10 3 -fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Å cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.The ever-growing trend among many bacterial pathogens toward antibiotic resistance represents one of the single greatest threats to public health in both developing and modern nations. In particular, a growing body of literature from the last decade has demonstrated that many strains of the widespread Gram-positive organisms Staphylococcus aureus and Staphylococcus epidermidis are now insensitive toward an array of the ␤-lactam class antibiotics that were once considered frontline therapeutics (1, 2). As recently as a few years ago, the problem of antibiotic resistance was associated primarily with those infections arising from within the healthcare setting. However, recent studies have shown that resistant strains are now spreading rapidly within the community, where they may cause potentially life-threatening illness in persons not recently hospitalized or undergoing invasive medical procedures (1, 3). Given the limited nature of effective therapeutic tools to combat these diseases, all such infections must be carefully managed to prevent further spread throughout the population. As a consequence, there is now renewed interest in novel classes of antimicrobials that are effective against sensitive and...

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“…Molecular-replacement searches were performed using this 133 amino-acid fragment of the 3fdu structure and using fragments of the other two closest homologues in the Protein Data Bank, represented by PDB entries 4lq8 (Lee et al, 2013) and 2hke (Byres et al, 2007). The molecular-replacement trials were carried out with MOLREP (Vagin & Teplyakov, 2010) and Phaser (McCoy et al, 2007), using different resolution limits and different tetragonal space groups.…”

Section: Resultsmentioning

confidence: 99%

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b fromPleurotus ostreatus

et al. 2013

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Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sittingdrop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris-HCl pH 8.5. The crystals belonged to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = 126.6, c = 53.9 Å . The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å .

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Crystal Structures of Trypanosoma brucei and Staphylococcus aureus Mevalonate Diphosphate Decarboxylase Inform on the Determinants of Specificity and Reactivity

Cited by 34 publications

References 55 publications

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b fromPleurotus ostreatus

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