31 research outputs found
Tetramerization reinforces the dimer interface of MnSOD.
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity
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Highly Active Yeast MnSOD has a Novel Mechanism Involving Six-coordinate Mn(3+) Species
Manganese-bound superoxide dismutase (MnSOD) is a very important antioxidant enzyme. The mechanism by which MnSOD removes O2— involves product inhibition, that is, reduction of O2— occurs through either a "prompt protonation" pathway, or an "inner-sphere" pathway, with the latter leading to formation of an observable Mn-peroxo complex. Human MnSOD is more gated toward the "inner-sphere" pathway than bacterial enzymes. To study whether product inhibition is a common feature to eukaryotic MnSODs, we studied a mitochondrial MnSOD from the eukaryote model organism Saccharomyces cerevisiae (ScMnSOD). To our surprise, ScMnSOD was found to display the highest catalytic efficiency at high levels of O2— among MnSODs that had been characterized. To understand further the mechanism of product inhibition, we compared ScMnSOD with another yeast MnSOD, the cytosolic MnSOD from Candida albicans (CaMnSODc). CaMnSODc, like ScMnSOD, is less inhibited than human and bacterial MnSODs. Although the active site of yeast MnSODs closely resembles that of MnSODs from other organisms, spectroscopic studies suggest the presence of a six-coordinate Mn3+ species in oxidized yeast MnSODs. To explore further the origin of the fast catalysis by yeast MnSODs, the Y34F (a strictly conserved second-sphere residue) form of ScMnSOD was created. Y34F ScMnSOD has a novel catalytic mechanism, in which protonation of the Mn-peroxo complex occurs through a fast pathway at neutral pH, leading to a putative six-coordinate Mn3+ species, which actively oxidizes O2— in the catalytic cycle. Because wild-type and the mutant yeast MnSOD both rest in the 2+ state and become six-coordinate when oxidized up from Mn2+, six-coordinate Mn3+ species could also actively function in the mechanism of wild-type yeast MnSODs. ScMnSOD is a tetramer, while CaMnSODc is a dimer or loose tetramer, even though they are similar in many ways. Investigations of their crystal structures suggest that when CaMnSODc is in the dimeric form, its N-terminal regions are highly disordered, hindering it from forming a tetramer in solution. To further investigate the physiological significance of the tetramer structure, we mutated two residues (Lys182/Ala183 in ScMnSOD, Lys184/Leu185 in CaMnSODc) at the dimer interface in the two yeast MnSODs. We find that the dimer interface, which is critical for MnSOD activity, is reinforced by tetramer formation
Exposure of Solvent-Inaccessible Regions in the Amyloidogenic Protein Human SOD1 Determined by Hydroxyl Radical Footprinting.
Highly Active Yeast MnSOD has a Novel Mechanism Involving Six-coordinate Mn(3+) Species
Manganese-bound superoxide dismutase (MnSOD) is a very important antioxidant enzyme. The mechanism by which MnSOD removes O2— involves product inhibition, that is, reduction of O2— occurs through either a "prompt protonation" pathway, or an "inner-sphere" pathway, with the latter leading to formation of an observable Mn-peroxo complex. Human MnSOD is more gated toward the "inner-sphere" pathway than bacterial enzymes. To study whether product inhibition is a common feature to eukaryotic MnSODs, we studied a mitochondrial MnSOD from the eukaryote model organism Saccharomyces cerevisiae (ScMnSOD). To our surprise, ScMnSOD was found to display the highest catalytic efficiency at high levels of O2— among MnSODs that had been characterized. To understand further the mechanism of product inhibition, we compared ScMnSOD with another yeast MnSOD, the cytosolic MnSOD from Candida albicans (CaMnSODc). CaMnSODc, like ScMnSOD, is less inhibited than human and bacterial MnSODs. Although the active site of yeast MnSODs closely resembles that of MnSODs from other organisms, spectroscopic studies suggest the presence of a six-coordinate Mn3+ species in oxidized yeast MnSODs. To explore further the origin of the fast catalysis by yeast MnSODs, the Y34F (a strictly conserved second-sphere residue) form of ScMnSOD was created. Y34F ScMnSOD has a novel catalytic mechanism, in which protonation of the Mn-peroxo complex occurs through a fast pathway at neutral pH, leading to a putative six-coordinate Mn3+ species, which actively oxidizes O2— in the catalytic cycle. Because wild-type and the mutant yeast MnSOD both rest in the 2+ state and become six-coordinate when oxidized up from Mn2+, six-coordinate Mn3+ species could also actively function in the mechanism of wild-type yeast MnSODs. ScMnSOD is a tetramer, while CaMnSODc is a dimer or loose tetramer, even though they are similar in many ways. Investigations of their crystal structures suggest that when CaMnSODc is in the dimeric form, its N-terminal regions are highly disordered, hindering it from forming a tetramer in solution. To further investigate the physiological significance of the tetramer structure, we mutated two residues (Lys182/Ala183 in ScMnSOD, Lys184/Leu185 in CaMnSODc) at the dimer interface in the two yeast MnSODs. We find that the dimer interface, which is critical for MnSOD activity, is reinforced by tetramer formation
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Exposure of Solvent-Inaccessible Regions in the Amyloidogenic Protein Human SOD1 Determined by Hydroxyl Radical Footprinting
Solvent-accessibility change plays a critical role in protein misfolding and aggregation, the culprit for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mass spectrometry-based hydroxyl radical (·OH) protein footprinting has evolved as a powerful and fast tool in elucidating protein solvent accessibility. In this work, we used fast photochemical oxidation of protein (FPOP) hydroxyl radical (·OH) footprinting to investigate solvent accessibility in human copper-zinc superoxide dismutase (SOD1), misfolded or aggregated forms of which underlie a portion of ALS cases. ·OH-mediated modifications to 56 residues were detected with locations largely as predicted based on X-ray crystallography data, while the interior of SOD1 β-barrel is hydrophobic and solvent-inaccessible and thus protected from modification. There were, however, two notable exceptions-two closely located residues inside the β-barrel, predicted to have minimal or no solvent accessibility, that were found modified by FPOP (Phe20 and Ile112). Molecular dynamics (MD) simulations were consistent with differential access of peroxide versus quencher to SOD1's interior complicating surface accessibility considerations. Modification of these two residues could potentially be explained either by local motions of the β-barrel that increased peroxide/solvent accessibility to the interior or by oxidative events within the interior that might include long-distance radical transfer to buried sites. Overall, comparison of modification patterns for the metal-free apoprotein versus zinc-bound forms demonstrated that binding of zinc protected the electrostatic loop and organized the copper-binding site. Our study highlights SOD1 hydrophobic groups that may contribute to early events in aggregation and discusses caveats to surface accessibility conclusions. Graphical Abstract
Comparative characterization of fungal anthracenone and naphthacenedione biosynthetic pathways reveals an a-hydroxylation-dependent claisen-like cyclization catalyzed by a dimanganese thioesterase
The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn2Cys6-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the α-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-β-lactamase-type thioesterase (MβL-TE). The bifunctional MβL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MβL-TE is a dimanganese enzyme and requires both Mn2+ cations for the observed activities. The MβL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/β hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi
Tetramerization reinforces the dimer interface of MnSOD.
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity
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Tetramerization reinforces the dimer interface of MnSOD.
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity