| 1. |
- Haddad Momeni, Majid, et al.
(författare)
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The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136, s. 321-329
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.
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| 2. |
- Johansson, Denny, 1980, et al.
(författare)
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Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:27, s. 9475-7
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Tidskriftsartikel (refereegranskat)abstract
- Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --> O or N --> S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.
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| 3. |
- Reetz, Manfred T., et al.
(författare)
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Directed Evolution of an Enantioselective Epoxide Hydrolase : Uncovering the Source of Enantioselectivity at Each Evolutionary Stage
- 2009
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Ingår i: Journal of the American Chemical Society. - : ACS. - 0002-7863 .- 1520-5126. ; 131:21, s. 7334-7343
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Forskningsöversikt (refereegranskat)abstract
- Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.
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| 4. |
- Sahin, Cagla, et al.
(författare)
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Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures
- 2023
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 145:19, s. 10659-10668
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Tidskriftsartikel (refereegranskat)abstract
- Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
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| 5. |
- Sandström, Corine
(författare)
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Mechanistic Investigations of Anaerobic Sulfatase-Maturating Enzyme: Direct C-beta H-Atom Abstraction Catalyzed by a Radical AdoMet Enzyme
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131, s. 8348-8349
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Tidskriftsartikel (refereegranskat)abstract
- Sulfatases are unique in requiring an essential post-translational modification of a critical active-site cysteinyt or seryl residue to 3-oxoalanine usually called C alpha-formylglycine (FGly). This post-translational modification is catalyzed anaerobically by anaerobic Sulfatase Maturating Enzyme (anSME), a member of the radical AdoMet superfamily. Using a new labeled substrate, we demonstrate that anSME uses a 5'-deoxyadenosyl radical to catalyze direct H-atom abstraction from the substrate. We thus established that anSMEs are the first radical AdoMet enzymes catalyzing a post-translational modification involving C, H-atom abstraction from an active site cysteinyl or seryl residue. This mechanistic study allowed us to decipher the first steps of the mechanism of this new radical AdoMet enzyme family.
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| 6. |
- Sjuvarsson, Elena, et al.
(författare)
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Nonpolar Nucleoside Mimics as Active Substrates for Human Thymidine Kinases
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131, s. 5488-5494
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Tidskriftsartikel (refereegranskat)abstract
- We describe the use of nonpolar nucleoside analogues of systematically varied size and shape to probe the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. Comparison of polar thymidine with a nonpolar isostere, 2,4-difluorotoluene deoxyriboside, as substrates for the two enzymes establishes that TK1 requires electrostatic complementarity to recognize the thymine base with high efficiency. Conversely, TK2 does not and phosphorylates the hydrophobic shape mimic with efficiency nearly the same as the natural substrate. To test the response to nucleobase size, thymidine-like analogues were systematically varied by replacing the 2,4 substituents on toluene with hydrogen and the halogen series (H, F, Cl, Br, I). Both enzymes showed a distinct preference for substrates having the natural size. To examine the shape preference, we prepared four mono- and difluorotoluene deoxyribosides with varying positions of substitutions. While TK1 did not accept these nonpolar analogues as substrates, TK2 did show varying levels of phosphorylation of the shape-varied set. This latter enzyme preferred toluene nucleoside analogues having steric projections at the 2 and 4 positions, as is found in thymine, and strongly disfavored substitution at the 3-position. Steady-state kinetics measurements showed that the 4-fluoro compound (7) had an apparent V(max)/K(m) value within 14-fold of the natural substrate, and the 2,4-difluoro compound (1), which is the closest isostere of thymidine, had a value within 2.5-fold. The results establish that nucleoside recognition mechanisms for the two classes of enzymes are very different. On the basis of these data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for future bioactive molecules.
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| 7. |
- Ståhlberg, Jerry
(författare)
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Carbohydrate-Protein Interactions That Drive Processive Polysaccharide Translocation in Enzymes Revealed from a Computational Study of Cellobiohydrolase Processivity
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136, s. 8810-8819
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Tidskriftsartikel (refereegranskat)abstract
- Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a glycoside hydrolase family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interactions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.
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| 8. |
- Uhlin, Elleonor
(författare)
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A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 132, s. 15368-15379
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli ribonucleotide reductase is an alpha 2 beta 2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y-center dot) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 angstrom between the two subunits by a specific pathway (Y-122(center dot)-> W-48 -> Y-356 within beta to Y-731 -> Y-730 -> C-439 within alpha). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO2Y) has recently been incorporated site-specifically in place of Y-122 in beta 2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO2Y center dot) and was inactive. In the present paper we show that incubation of apo-Y122NO2Y-beta 2 with Fe2+ and O-2 generates a diferric-NO2Y center dot that has a half-life of 40 s at 25 degrees C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO2Y center dot/beta 2 and then mixed with alpha 2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO2Y center dot is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y-center dot is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y-center dot is predominantly located at 356 in beta 2. In consideration of these data and the crystal structure of Y122NO2Y-beta 2, a mechanism for PCET uncoupling in NO2Y center dot-RNR is proposed.
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| 9. |
- Uhlin, Ulla
(författare)
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Kinetics of Radical Intermediate Formation and Deoxynucleotide Production in 3-Aminotyrosine-Substituted Escherichia coli Ribonucleotide Reductases
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 133, s. 9430-9440
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli ribonucleotide reductase is an alpha 2 beta 2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in alpha 2 by a stable diferric tyrosyl radical (Y(122)center dot) cofactor in beta 2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 A through a specific pathway of residues: Y(122)center dot -> W(48)-> Y(356) in beta 2 to Y(731) -> Y(730) -> C(439) in alpha 2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of beta. The resulting protein, Y(336)NH(2)Y-beta 2, and the previously generated proteins Y(731)NH(2)Y-alpha 2 and Y(730)NH(2)Y-alpha 2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (5), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y center dot) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y center dot formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH2Y center dot formation, while the k(cat) of similar to 0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y center dot of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-alpha 2 and Y(731)NH(2)Y-alpha 2 have been solved and indicate minimal structural changes relative to wt-alpha 2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.
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| 10. |
- Uhlin, Ulla
(författare)
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Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK(a) Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 132, s. 8385-8397
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Tidskriftsartikel (refereegranskat)abstract
- E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5'-diphosphates into 2'-deoxynucleotides and is composed of two subunits: alpha 2 and beta 2. During turnover, a stable tyrosyl radical (Y center dot) at Y-122-beta 2 reversibly oxidizes C-439 in the active site of alpha 2. This radical propagation step is proposed to occur over 35 angstrom, to use specific redox-active tyrosines (Y-122 and Y-356 in beta 2, Y-731 and Y-730 in alpha 2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO2Y, pK(a) 7.1) has been incorporated in place of Y-122, Y-731, and Y-730 to probe how the protein environment perturbs each pK(a) in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 x 10(-3) that of the wild-type (wt) subunit. The [NO2Y730]-alpha 2 and [NO2Y731]-alpha 2 each exhibit a pKa of 7.8-8.0 with E and E/beta 2. The pK(a) of [NO2Y730]-alpha 2 is elevated to 8.2-8.3 in the S/E/beta 2 complex, whereas no further perturbation is observed for [NO2Y731]-alpha 2. Mutations in pathway residues adjacent to the NO2Y that disrupt H-bonding minimally perturb its pK(a). The pK(a) of NO2Y122-beta 2 alone or with alpha 2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO2Y]-alpha 2 mutants (2.1-3.1 angstrom resolution), which show minimal structural perturbation compared to wt-alpha 2. Together with the pK(a) of the previously reported NO2Y356-beta 2 (7.5 in the alpha 2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541-14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway.
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