| 1. |
- Baier, Florian, et al.
(författare)
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Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes
- 2019
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-beta-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.
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| 2. |
- Burke, Jason R., et al.
(författare)
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Bifunctional Substrate Activation via an Arginine Residue Drives Catalysis in Chalcone Isomerases
- 2019
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 9:9, s. 8388-8396
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Tidskriftsartikel (refereegranskat)abstract
- Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex combined with molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Bronsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.
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| 3. |
- Calixto, Ana R., et al.
(författare)
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GTP Hydrolysis Without an Active Site Base : A Unifying Mechanism for Ras and Related GTPases
- 2019
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Ingår i: Journal of the American Chemical Society. - : AMER CHEMICAL SOC. - 0002-7863 .- 1520-5126. ; 141:27, s. 10684-10701
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Tidskriftsartikel (refereegranskat)abstract
- GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the G(alpha i), subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.
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| 4. |
- Kaltenbach, Miriam, et al.
(författare)
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Evolution of chalcone isomerase from a noncatalytic ancestor
- 2018
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Ingår i: Nature Chemical Biology. - : NATURE PUBLISHING GROUP. - 1552-4450 .- 1552-4469. ; 14:6, s. 548-555
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substratebinding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.
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| 5. |
- Liao, Qinghua, et al.
(författare)
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Extending the Nonbonded Cationic Dummy Model to Account for Ion-Induced Dipole Interactions
- 2017
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 8:21, s. 5408-5414
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Tidskriftsartikel (refereegranskat)abstract
- Modeling metalloproteins often requires classical molecular dynamics (MD) simulations in order to capture their relevant motions, which in turn necessitates reliable descriptions of the metal centers involved. One of the most successful approaches to date is provided by the "cationic dummy model", where the positive charge of the metal ion is transferred toward dummy particles that are bonded to the central metal ion in a predefined coordination geometry. While this approach allows for ligand exchange, and captures the correct electrostatics as demonstrated for different divalent metal ions, current dummy models neglect ion-induced dipole interactions. In the present work, we resolve this weakness by taking advantage of the recently introduced 12-6-4 type Lennard-Jones potential to include ion-induced dipole interactions. We revise our previous dummy model for Mg2+ and demonstrate that the resulting model can simultaneously reproduce the experimental solvation free energy and metal ligand distances without the need for artificial restraints or bonds. As ion-induced dipole interactions become particularly important for highly charged metal ions, we develop dummy models for the biologically relevant ions Al3+, Fe3+, and Cr3+. Finally, the effectiveness of our new models is demonstrated in MD simulations of several diverse (and highly challenging to simulate) metalloproteins.
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| 6. |
- Pabis, Anna, et al.
(författare)
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Cooperativity and flexibility in enzyme evolution
- 2018
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Ingår i: Current opinion in structural biology. - : CURRENT BIOLOGY LTD. - 0959-440X .- 1879-033X. ; 48, s. 83-92
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.
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| 7. |
- Pabis, Anna, et al.
(författare)
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Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:13, s. 7200-7207
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Tidskriftsartikel (refereegranskat)abstract
- Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intra-membrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring before stalk formation and one after, are consistent with our experimental measurements of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate previous mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also use sequential processes of acyl tail exposure, followed by membrane curvature and distal leaflet engagement.
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| 8. |
- Pabis, Anna, et al.
(författare)
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Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily
- 2016
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Ingår i: Current opinion in structural biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; 37, s. 14-21
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Tidskriftsartikel (refereegranskat)abstract
- Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity.
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| 9. |
- Pabis, Anna, et al.
(författare)
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Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:22, s. 3061-3081
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Tidskriftsartikel (refereegranskat)abstract
- The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer.
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| 10. |
- Pabis, Anna, et al.
(författare)
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Simulating the reactions of substituted pyridinio-N-phosphonates with pyridine as a model for biological phosphoryl transfer
- 2017
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 15:35, s. 7308-7316
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoryl transfer reactions can proceed through several plausible mechanisms, and the potential for both solvent and substrate-assisted pathways (involving proton transfer to the phosphoryl oxygens) complicates both experimental and computational interpretations. To avoid this problem, we have used electronic structure calculations to probe the mechanisms of the reactions of pyridinio-N-phosphonates with pyridine. These compounds avoid the additional complexity introduced by proton transfer between the nucleophile and the leaving group, while also serving as a valuable model for biological P-N cleavage. Through a comparative study of a range of substrates of varying basicity, we demonstrate a unified concerted mechanism for the phosphoryl transfer reactions of these model compounds, proceeding through a dissociative transition state. Finally, a comparison of these transition states with previously characterized transition states for related compounds provides a more complete model for non-enzymatic phosphoryl transfer, which is a critical stepping stone to being able to fully understand phosphoryl transfer in biology.
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| 11. |
- Pati, Sarah G., et al.
(författare)
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Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants
- 2016
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Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 50:13, s. 6708-6716
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Tidskriftsartikel (refereegranskat)abstract
- Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent C-13- and H-2-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O-2 activation and aromatic C oxygenation. The contribution of enzymatic O-2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O-2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.
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| 12. |
- Purg, Miha, et al.
(författare)
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Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase.
- 2016
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Ingår i: Philosophical Transactions of the Royal Society of London A, Mathematical, Physical and Engineering Sciences. - : The Royal Society. ; 374, s. 20160150-
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Tidskriftsartikel (refereegranskat)abstract
- Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal–metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’.
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| 13. |
- Uduwela, Dimanthi R., et al.
(författare)
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Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa
- 2018
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 8:9, s. 8902-8914
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Tidskriftsartikel (refereegranskat)abstract
- Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolyzing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semirational design to mutate amino acid residues neighboring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by ultra-high performance liquid chromatography-mass spectrometry was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (V-max/K-M) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded, and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects.
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