RESUMO
Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate. Herein we pursue the time and temperature dependence of solvation dynamics at the surface of mADA, for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with close to native kinetic behavior. Single site-specific tryptophan mutants were back inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays, that reflect time dependent environmental reoroganization around the photo-excited state of Trp*, display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent exposed region near the helix-loop-helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for Ea ( k Stokes shift ) ; further, when Phe61 within the long helix positioned behind bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in Ea ( k Stokes shift ) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, Ea ( k cat ) .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.
RESUMO
Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein-protein and protein-peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.
RESUMO
The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.
Assuntos
Glycine max , Lipoxigenase , Corantes Fluorescentes , Movimento (Física) , HidrogênioRESUMO
Formulations of hydrogen tunneling in enzyme-catalysed C-H activation reactions indicate enthalpic barriers to reaction that are independent of chemical steps and dependent on the protein scaffold. A tool to identify catalytically relevant site-specific protein thermal networks has emerged from temperature-dependent hydrogen deuterium exchange (TDHDX). Focusing on mutant enzyme forms with altered activation energies for catalysis, TDHDX provides a comparative analysis of the impact of mutation on Ea for local protein unfolding. Identified thermal networks appear unrelated to protein scaffold conservation and track to the dictates of the catalysed reaction, including sites for metal binding. The positions of thermal networks provide a framework for further understanding of time-dependent, functionally relevant protein motions. Measurement of nanosecond Stokes shifts at the surface of the thermal network in soybean lipoxygenase yields activation energies that are identical to Ea values measured for kcat . This finding identifies a rapid (> nanosecond), long-range and cooperative structural reorganization as the thermal barrier to catalysis. A model for protein dynamics is put forward that integrates broadly distributed protein conformational sampling with protein embedded thermal networks.
Assuntos
Hidrogênio , Proteínas , Modelos Moleculares , Hidrogênio/química , Termodinâmica , Temperatura , Catálise , Lipoxigenase/genética , Lipoxigenase/química , Lipoxigenase/metabolismo , CinéticaRESUMO
The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 106-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.
Assuntos
Adenosina Desaminase , Deutério , Adenosina/química , Adenosina Desaminase/química , Deutério/química , Medição da Troca de Deutério , Ligantes , Pentostatina/química , Conformação Proteica , Proteínas , TemperaturaRESUMO
With the growing acceptance of the contribution of protein conformational ensembles to enzyme catalysis and regulation, research in the field of protein dynamics has shifted toward an understanding of the atomistic properties of protein dynamical networks and the mechanisms and time scales that control such behavior. A full description of an enzymatic reaction coordinate is expected to extend beyond the active site and include site-specific networks that communicate with the protein/water interface. Advances in experimental tools for the spatial resolution of thermal activation pathways are being complemented by biophysical methods for visualizing dynamics in real time. An emerging multidimensional model integrates the impacts of bound substrate/effector on the distribution of protein substates that are in rapid equilibration near room temperature with reaction-specific protein embedded heat transfer conduits.
Assuntos
Proteínas , Biofísica , Catálise , Domínio Catalítico , Conformação Proteica , Proteínas/químicaRESUMO
We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pKa of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein-solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.
Assuntos
Liases/metabolismo , Temperatura , Água/metabolismo , Sítios de Ligação , Biocatálise , Cinética , Liases/química , Liases/genética , Modelos Moleculares , Mutação , Saccharomyces cerevisiae/enzimologia , Água/químicaRESUMO
Proteins are intrinsically flexible macromolecules that undergo internal motions with time scales spanning femtoseconds to milliseconds. These fluctuations are implicated in the optimization of reaction barriers for enzyme catalyzed reactions. Time, temperature, and mutation dependent hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been previously employed to identify spatially resolved, catalysis-linked dynamical regions of enzymes. We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure-function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 7.5 kcal/mol) and the wild type (WT) mADA (Ea = 5.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for kcat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.
Assuntos
Adenosina Desaminase/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério , Adenosina/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/genética , Animais , Sítios de Ligação , Biocatálise , Desaminação , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Especificidade por Substrato , TemperaturaRESUMO
Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.
Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Ferro/metabolismo , Methylobacterium extorquens/química , Enxofre/metabolismo , Proteínas de Bactérias/química , Biocatálise , Cristalografia por Raios X , Endopeptidases/química , Ferro/química , Methylobacterium extorquens/metabolismo , Modelos Moleculares , Estrutura Molecular , Enxofre/químicaRESUMO
Pyrroloquinoline quinone (PQQ) is a peptide-derived redox cofactor produced by prokaryotes that also plays beneficial roles in organisms from other kingdoms. We review recent developments on the pathway of PQQ biogenesis, focusing on the mechanisms of PqqE, PqqF/G, and PqqB. These advances may shed light on other, uncharacterized biosynthetic pathways.
Assuntos
Bactérias/metabolismo , Vias Biossintéticas , Cofator PQQ/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Moleculares , OxirreduçãoRESUMO
Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954-7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20 °C indicates changes in flexibility across â¼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.
Assuntos
Catecol O-Metiltransferase/química , Motivos de Aminoácidos , Domínio Catalítico , Catecol O-Metiltransferase/genética , Catecol O-Metiltransferase/metabolismo , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Simulação de Dinâmica Molecular , MutaçãoRESUMO
Hydrogen tunneling in enzymatic C-H activation requires a dynamical sampling among ground-state enzyme-substrate (E-S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor-acceptor distance (DAD) of 2.7 Å, â¼0.5 Å shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13C electron-nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme-substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)-OH]: the active conformer "a", with a van der Waals DAD of 3.1 Å between C11 and metal-bound hydroxide, and an inactive conformer "b", with a distance that is almost 1 Å longer. Herein, the structure of the E-S complex is examined for a series of six variants in which subtle structural modifications of SLO have been introduced either at a hydrophobic side chain near the bound substrate or at a remote residue within a protein network whose flexibility influences hydrogen transfer. A remarkable correlation is found between the ENDOR-derived population of the active ground-state conformer a and the kinetically derived differential enthalpic barrier for D versus H transfer, ΔEa, with the latter increasing as the fraction of conformer a decreases. As proposed, ΔEa provides a "ruler" for the DAD within the TRS. ENDOR measurements further corroborate the previous identification of a dynamical network coupling the buried active site of SLO to the surface. This study shows that subtle imperfections within the initial ground-state structures of E-S complexes are accompanied by compromised geometries at the TRS.
Assuntos
Glycine max/enzimologia , Ácido Linoleico/química , Lipoxigenase/química , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Hidrogênio/química , Lipoxigenase/genética , Mutação , Conformação ProteicaRESUMO
Pyrroloquinoline quinone (PQQ) is an important redox active quinocofactor produced by a wide variety of bacteria. A key step in PQQ biosynthesis is a carbon-carbon cross-link reaction between glutamate and tyrosine side chains within the ribosomally synthesized peptide substrate PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous X-ray crystallographic and spectroscopic studies suggested that PqqE, like the other members of the SPASM domain family, contains two auxiliary Fe-S clusters (AuxI and AuxII) in addition to the radical SAM [4Fe-4S] cluster. However, a clear assignment of the electron paramagnetic resonance (EPR) signal of each Fe-S cluster was hindered by the isolation of a His6-tagged PqqE variant with an altered AuxI cluster. In this work, we are able to isolate soluble PqqE variants by using a less disruptive strep-tactin chromatographic approach. We have unambiguously identified the EPR signatures for four forms of Fe-S clusters present in PqqE through the use of multifrequency EPR spectroscopy: the RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and two different clusters ([4Fe-4S] and [2Fe-2S]) bound in the AuxI site. The RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and the [2Fe-2S] cluster form in the AuxI site can all be reduced by sodium dithionite, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940, 1.903], and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe-4S] cluster that is determined on the basis of its relaxation profile can be reduced only by using low-potential reductants such as Ti(III) citrate or Eu(II)-DTPA to give rise to a g1 = 2.104 signal. Identification of the EPR signature for each cluster paves the way for further investigations of SPASM domain radical SAM enzymes.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Endopeptidases/química , Endopeptidases/metabolismo , Ferro , Enxofre , Biocatálise , Cofator PQQ/metabolismo , Domínios ProteicosRESUMO
The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317-334) that correlate well with trends in the corresponding enthalpic barriers for kcat. A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD (Ea(ksolv) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation (Ea(kcat) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced "protein quakes" as the origin of enthalpic barriers in catalysis.
Assuntos
Glycine max/enzimologia , Lipoxigenase/química , Lipoxigenase/metabolismo , Solventes/química , Sítios de Ligação , Catálise , Domínio Catalítico , Cinética , Modelos Moleculares , Conformação Proteica , Temperatura , TermodinâmicaRESUMO
Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate.
Assuntos
Glycine max/enzimologia , Lipoxigenase/química , Proteínas de Soja/química , Substituição de Aminoácidos , Domínio Catalítico , Lipoxigenase/genética , Mutação de Sentido Incorreto , Proteínas de Soja/genética , Glycine max/genéticaRESUMO
Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB-E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the α-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a KD of 300 nm We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.
Assuntos
Alphaproteobacteria/enzimologia , Coenzimas/metabolismo , Cofator PQQ/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Oxirredução , Peptídeo Hidrolases/química , Ligação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de ProteínaRESUMO
This first serious attempt at an autobiographical accounting has forced me to sit still long enough to compile my thoughts about a long personal and scientific journey. I especially hope that my trajectory will be of interest and perhaps beneficial to much younger women who are just getting started in their careers. To paraphrase from Virginia Woolf's writings in A Room of One's Own at the beginning of the 20th century, "for most of history Anonymous was a Woman." However, Ms. Woolf is also quoted as saying "nothing has really happened until it has been described," a harbinger of the enormous historical changes that were about to be enacted and recorded by women in the sciences and other disciplines. The progress in my chosen field of study-the chemical basis of enzyme action-has also been remarkable, from the first description of an enzyme's 3D structure to a growing and deep understanding of the origins of enzyme catalysis.
Assuntos
Coenzimas/química , Enzimas/química , Mulheres Trabalhadoras/história , Biocatálise , Escolha da Profissão , Coenzimas/metabolismo , Ensaios Enzimáticos , Enzimas/metabolismo , Feminino , História do Século XX , História do Século XXI , Humanos , Cinética , Teoria QuânticaRESUMO
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de HidrogênioRESUMO
Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.