RESUMO
How individual enzymes evolved is relatively well understood. However, individual enzymes rarely confer a physiological advantage on their own. Judging by its current state, the emergence of metabolism seemingly demanded the simultaneous emergence of many enzymes. Indeed, how multicomponent interlocked systems, like metabolic pathways, evolved is largely an open question. This complexity can be unlocked if we assume that survival of the fittest applies not only to genes and enzymes but also to the metabolites they produce. This review develops our current knowledge of enzyme evolution into a wider hypothesis of pathway and network evolution. We describe the current models for pathway evolution and offer an integrative metabolite-enzyme coevolution hypothesis. Our hypothesis addresses the origins of new metabolites and of new enzymes and the order of their recruitment. We aim to not only survey established knowledge but also present open questions and potential ways of addressing them.
Assuntos
Enzimas/genética , Enzimas/metabolismo , Evolução Molecular , Redes e Vias Metabólicas/genética , Enzimas/química , Cinética , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Filogenia , Especificidade por Substrato/genéticaRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.
Assuntos
DNA/genética , Escherichia coli/genética , Recombinação Genética , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Aberrações Cromossômicas , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/metabolismo , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de MoléculaRESUMO
Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.
Assuntos
Proteínas Argonautas , Thermus thermophilus , Proteínas Argonautas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/genética , Endonucleases/metabolismo , Thermus thermophilus/genéticaRESUMO
Phase separation can concentrate biomolecules and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects associated with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concentration within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromolecular crowding. Finally, the phosphorylation of the Alzheimer's-disease-associated protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophysical properties of the cytoplasm.
Assuntos
Transdução de Sinais , Proteínas tau , Quinase 2 Dependente de Ciclina/metabolismo , Citoplasma/metabolismo , Humanos , Substâncias Macromoleculares/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Multicellular organisms develop complex shapes from much simpler, single-celled zygotes through a process commonly called morphogenesis. Morphogenesis involves an interplay between several factors, ranging from the gene regulatory networks determining cell fate and differentiation to the mechanical processes underlying cell and tissue shape changes. Thus, the study of morphogenesis has historically been based on multidisciplinary approaches at the interface of biology with physics and mathematics. Recent technological advances have further improved our ability to study morphogenesis by bridging the gap between the genetic and biophysical factors through the development of new tools for visualizing, analyzing, and perturbing these factors and their biochemical intermediaries. Here, we review how a combination of genetic, microscopic, biophysical, and biochemical approaches has aided our attempts to understand morphogenesis and discuss potential approaches that may be beneficial to such an inquiry in the future.
Assuntos
Morfogênese , Biofísica , Diferenciação Celular , Morfogênese/genéticaRESUMO
The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína Axina/genética , beta Catenina/genética , Proteína da Polipose Adenomatosa do Colo/ultraestrutura , Proteína Axina/química , Proteína Axina/ultraestrutura , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Fosforilação/genética , Multimerização Proteica/genética , Proteólise , Ubiquitinação/genética , Via de Sinalização WntRESUMO
Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Imunidade Adaptativa/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Cruciforme , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteína 1 Homóloga a MutL/genéticaRESUMO
The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.
Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Proteínas Relacionadas à Autofagia , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genéticaRESUMO
The potential of engineered enzymes in industrial applications is often limited by their expression levels, thermal stability, and catalytic diversity. De novo enzyme design faces challenges due to the complexity of enzymatic catalysis. An alternative approach involves expanding natural enzyme capabilities for new substrates and parameters. Here, we introduce CoSaNN (Conformation Sampling using Neural Network), an enzyme design strategy using deep learning for structure prediction and sequence optimization. CoSaNN controls enzyme conformations to expand chemical space beyond simple mutagenesis. It employs a context-dependent approach for generating enzyme designs, considering non-linear relationships in sequence and structure space. We also developed SolvIT, a graph NN predicting protein solubility in Escherichia coli, optimizing enzyme expression selection from larger design sets. Using this method, we engineered enzymes with superior expression levels, with 54% expressed in E. coli, and increased thermal stability, with over 30% having higher Tm than the template, with no high-throughput screening. Our research underscores AI's transformative role in protein design, capturing high-order interactions and preserving allosteric mechanisms in extensively modified enzymes, and notably enhancing expression success rates. This method's ease of use and efficiency streamlines enzyme design, opening broad avenues for biotechnological applications and broadening field accessibility.
Assuntos
Aprendizado Profundo , Escherichia coli/genética , Biotecnologia , Catálise , Ensaios de Triagem em Larga EscalaRESUMO
Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.
Assuntos
Gafanhotos , Toxinas Biológicas , Sistemas de Secreção Tipo VII , Animais , Sistemas de Secreção Tipo VII/genética , CitoplasmaRESUMO
The stationary phase promoter specificity subunit σS (RpoS) is delivered to the ClpXP machinery for degradation dependent on the adaptor RssB. This adaptor-specific degradation of σS provides a major point for regulation and transcriptional reprogramming during the general stress response. RssB is an atypical response regulator and the only known ClpXP adaptor that is inhibited by multiple but dissimilar antiadaptors (IraD, IraP, and IraM). These are induced by distinct stress signals and bind to RssB in poorly understood manners to achieve stress-specific inhibition of σS turnover. Here we present the first crystal structure of RssB bound to an antiadaptor, the DNA damage-inducible IraD. The structure reveals that RssB adopts a compact closed architecture with extensive interactions between its N-terminal and C-terminal domains. The structural data, together with mechanistic studies, suggest that RssB plasticity, conferred by an interdomain glutamate-rich flexible linker, is critical for regulation of σS degradation. Structural modulation of interdomain linkers may thus constitute a general strategy for tuning response regulators.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Fator sigma/química , Fator sigma/metabolismo , Fatores de Transcrição/química , Proteínas de Bactérias/química , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Estabilidade Proteica , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Microbiology began as a unified science using the principles of chemistry to understand living systems. The unified view quickly split into the subdisciplines of medical microbiology, molecular biology, and environmental microbiology. The advent of a universal phylogeny and culture-independent approaches has helped tear down the boundaries separating the subdisciplines. The vision for the future is that the study of the fundamental roles of microbes in ecology and evolution will lead to an integrated biology with no boundary between microbiology and macrobiology.
Assuntos
Ecologia , Biologia Molecular , Microbiologia , FilogeniaRESUMO
Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas , Código das Histonas , Ubiquitinação , LógicaRESUMO
Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
Assuntos
Nanopartículas , Vacinas , Proteínas , Nanopartículas/químicaRESUMO
Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.
Assuntos
Dobramento de RNA , RNA , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA/metabolismo , TermodinâmicaRESUMO
Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.
Assuntos
Alphavirus , Febre de Chikungunya , Alphavirus/genética , Antivirais/farmacologia , Guanosina Trifosfato/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme's conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme's conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.
Assuntos
Monoéster Fosfórico Hidrolases , Compostos de Tungstênio , Catálise , Mutação , Cinética , Sítios de LigaçãoRESUMO
Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.