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1.
Mol Cell ; 72(1): 178-186.e5, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30270109

RESUMO

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.


Assuntos
Domínio Catalítico , Coenzima A Ligases/química , Hidrolases de Triester Fosfórico/química , Engenharia de Proteínas , Acil Coenzima A/biossíntese , Acil Coenzima A/química , Catálise , Coenzima A Ligases/genética , Cinética , Mutação , Compostos Organofosforados/química , Hidrolases de Triester Fosfórico/genética , Filogenia , Software , Especificidade por Substrato
2.
Annu Rev Biochem ; 79: 471-505, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20235827

RESUMO

Many, if not most, enzymes can promiscuously catalyze reactions, or act on substrates, other than those for which they evolved. Here, we discuss the structural, mechanistic, and evolutionary implications of this manifestation of infidelity of molecular recognition. We define promiscuity and related phenomena and also address their generality and physiological implications. We discuss the mechanistic enzymology of promiscuity--how enzymes, which generally exert exquisite specificity, catalyze other, and sometimes barely related, reactions. Finally, we address the hypothesis that promiscuous enzymatic activities serve as evolutionary starting points and highlight the unique evolutionary features of promiscuous enzyme functions.


Assuntos
Enzimas/genética , Catálise , Domínio Catalítico , Enzimas/química , Enzimas/metabolismo , Evolução Molecular , Humanos , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723058

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.


Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Oxirredutases N-Desmetilantes/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Tobramicina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Oxirredutases N-Desmetilantes/farmacologia , Engenharia de Proteínas , Pseudomonas aeruginosa/fisiologia
4.
Proc Natl Acad Sci U S A ; 115(49): E11455-E11464, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30459276

RESUMO

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2 Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2 Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic-stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco's maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.


Assuntos
Dióxido de Carbono/metabolismo , Glicolatos/metabolismo , Fotossíntese/fisiologia , Simulação por Computador , Engenharia Metabólica , Modelos Biológicos , Engenharia de Proteínas , Ribulose-Bifosfato Carboxilase/metabolismo , Biologia Sintética
5.
Proc Natl Acad Sci U S A ; 109(26): 10358-63, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22685214

RESUMO

Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K(m) values of ≤ 5 10(4) M(-1)s(-1)). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k(cat)/K(m), and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K(m) values up to 0.6 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of ≤ 10(7) almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.


Assuntos
Evolução Molecular Direcionada , Enzimas/metabolismo , Domínio Catalítico , Estabilidade Enzimática
6.
Nature ; 453(7192): 190-5, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18354394

RESUMO

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.


Assuntos
Simulação por Computador , Evolução Molecular Direcionada/métodos , Enzimas/química , Enzimas/metabolismo , Engenharia de Proteínas/métodos , Algoritmos , Motivos de Aminoácidos , Sítios de Ligação/genética , Catálise , Biologia Computacional , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Enzimas/genética , Cinética , Modelos Químicos , Modelos Moleculares , Teoria Quântica , Sensibilidade e Especificidade
7.
Nat Genet ; 37(1): 73-6, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15568024

RESUMO

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.


Assuntos
Arildialquilfosfatase/genética , Anidrase Carbônica II/genética , Evolução Molecular , Hidrolases de Triester Fosfórico/genética , Arildialquilfosfatase/fisiologia , Bactérias/enzimologia , Bactérias/genética , Anidrase Carbônica II/fisiologia , Variação Genética , Humanos , Hidrolases de Triester Fosfórico/fisiologia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína
8.
J Mol Biol ; 435(17): 168191, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37385581

RESUMO

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.


Assuntos
Proteínas Recombinantes , Albumina Sérica , Animais , Humanos , Dissulfetos , Escherichia coli/genética , Reprodutibilidade dos Testes , Albumina Sérica/genética , Albumina Sérica/química , Albumina Sérica Humana/química , Albumina Sérica Humana/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
FEBS J ; 290(13): 3383-3399, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36808692

RESUMO

Acid-ß-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.


Assuntos
Celulases , Doença de Gaucher , Doença de Parkinson , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Parkinson/genética , Heterozigoto , Mutação , Celulases/genética
10.
Biodes Res ; 2022: 9787581, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37850148

RESUMO

The overarching goal of computational protein design is to gain complete control over protein structure and function. The majority of sophisticated binders and enzymes, however, are large and exhibit diverse and complex folds that defy atomistic design calculations. Encouragingly, recent strategies that combine evolutionary constraints from natural homologs with atomistic calculations have significantly improved design accuracy. In these approaches, evolutionary constraints mitigate the risk from misfolding and aggregation, focusing atomistic design calculations on a small but highly enriched sequence subspace. Such methods have dramatically optimized diverse proteins, including vaccine immunogens, enzymes for sustainable chemistry, and proteins with therapeutic potential. The new generation of deep learning-based ab initio structure predictors can be combined with these methods to extend the scope of protein design, in principle, to any natural protein of known sequence. We envision that protein engineering will come to rely on completely computational methods to efficiently discover and optimize biomolecular activities.

11.
Nat Commun ; 13(1): 3023, 2022 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-35641498

RESUMO

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Engenharia Genética , Genoma , Mamíferos
12.
Nat Biotechnol ; 40(7): 1143-1149, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35102291

RESUMO

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.


Assuntos
Imageamento por Ressonância Magnética , Animais , Genes Reporter/genética , Imageamento por Ressonância Magnética/métodos , Transgenes
13.
Biochemistry ; 50(13): 2683-90, 2011 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-21332126

RESUMO

Two different scenarios for the recruitment of evolutionary starting points and their subsequent divergence to give new enzymes have been described. The coincidental, promiscuous starting activity may regard the same reaction chemistry on a new substrate (substrate ambiguity). Alternatively, substrate binding guides the recruitment of an enzyme whose reaction chemistry differs from that of the newly evolving one (catalytic promiscuity). While substrate ambiguity seems to underlie the divergence of most enzyme families, the relative levels of occurrence of these scenarios remain unknown. Screening the Escherichia coli proteome with a comparative series of xenobiotic substrates, we found that substrate ambiguity was, as anticipated, more frequent than reaction promiscuity. However, for at least one unnatural reaction (phosphonoesterase), a promiscuous enzyme was identified only when the substrate was decorated with the naturally abundant phosphate group. These findings support the prevailing hypothesis of chemistry-driven divergence but also suggest that recognition of familiar substrate motifs plays a role. In the absence of enzymes catalyzing the same chemistry, having a familiar, naturally occurring substrate motif (chemophore) such as phosphate may increase the likelihood of catalytic promiscuity. Chemophore anchoring may also find practical applications in identifying catalysts for unnatural reactions.


Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Proteoma/metabolismo , Xenobióticos/metabolismo , Biocatálise , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Especificidade por Substrato , Xenobióticos/química
14.
ACS Sens ; 6(11): 4193-4205, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34783546

RESUMO

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish diverse functions in small molecule transport, sensing, and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 ± 0.5 µM), specificity (40-fold increase vs glycine), thermostability (Tm = 79 °C), and dynamic range (∼14%). This sensor allowed measurement of physiologically relevant changes in d-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity, and thermostability and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Animais , Sítios de Ligação , Ligantes , Ratos , Serina
15.
Curr Opin Struct Biol ; 63: 58-64, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32505941

RESUMO

Our ability to design new or improved biomolecular activities depends on understanding the sequence-function relationships in proteins. The large size and fold complexity of most proteins, however, obscure these relationships, and protein-optimization methods continue to rely on laborious experimental iterations. Recently, a deeper understanding of the roles of stability-threshold effects and biomolecular epistasis in proteins has led to the development of hybrid methods that combine phylogenetic analysis with atomistic design calculations. These methods enable reliable and even single-step optimization of protein stability, expressibility, and activity in proteins that were considered outside the scope of computational design. Furthermore, ancestral-sequence reconstruction produces insights on missing links in the evolution of enzymes and binders that may be used in protein design. Through the combination of phylogenetic and atomistic calculations, the long-standing goal of general computational methods that can be universally applied to study and optimize proteins finally seems within reach.


Assuntos
Biologia Computacional/métodos , Filogenia , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Automação , Ligantes , Modelos Moleculares , Mutagênese , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
16.
Biochemistry ; 48(28): 6644-54, 2009 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-19492856

RESUMO

Serum paraoxonases (PONs) are calcium-dependent lactonases with anti-atherogenic and detoxification functions. Here we describe the directed evolution and characterization of recombinant variants of serum paraoxonase PON3 that express in an active and soluble manner in Escherichia coli. These variants were obtained by combining family shuffling and phylogeny-based mutagenesis: the limited diversity of accessible, cloned PON3 genes was complemented by spiking the shuffling reaction with ancestor/consensus mutations, mutations to residues that comprise the consensus or appear in the predicted ancestors of the PON family. We screened the resulting libraries for PON3's lactonase activity while ensuring that the selected variants retained the substrate specificity of wild-type mammalian PON3s. The availability of highly stable, recombinant PON3 that is free of all other serum components enabled us to explore unknown biochemical features of PON3, including its binding to HDL particles, the effect of HDL on PON3's stability and enzymatic activity, and ex vivo tests of its anti-atherogenic properties. Overall, it appears that PON3 possesses properties very similar to those of PON1: the enzyme's lactonase activity is selectively stimulated by binding to apoAI-HDL, with a concomitant increase in its stability. PON3 also exhibits potentially anti-atherogenic functions, although at levels lower than those of PON1.


Assuntos
Arildialquilfosfatase/sangue , Sequência Consenso , Embaralhamento de DNA , Evolução Molecular Direcionada , Mutagênese , Sequência de Aminoácidos , Animais , Arildialquilfosfatase/química , Aterosclerose/enzimologia , Biocatálise , Extratos Celulares , Colesterol/metabolismo , Ativação Enzimática , Escherichia coli , Humanos , Cinética , Lipoproteínas HDL , Macrófagos/enzimologia , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/sangue , Proteínas Mutantes/química , Oxirredução , Estabilidade Proteica , Coelhos , Análise de Sequência de Proteína , Especificidade por Substrato
17.
Nat Struct Mol Biol ; 11(5): 412-9, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15098021

RESUMO

Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.


Assuntos
Arildialquilfosfatase/sangue , Arildialquilfosfatase/genética , Evolução Molecular , Sequência de Aminoácidos , Arildialquilfosfatase/química , Arildialquilfosfatase/metabolismo , Catálise , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
18.
Nat Commun ; 9(1): 2780, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018322

RESUMO

Automated design of enzymes with wild-type-like catalytic properties has been a long-standing but elusive goal. Here, we present a general, automated method for enzyme design through combinatorial backbone assembly. Starting from a set of homologous yet structurally diverse enzyme structures, the method assembles new backbone combinations and uses Rosetta to optimize the amino acid sequence, while conserving key catalytic residues. We apply this method to two unrelated enzyme families with TIM-barrel folds, glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase-like lactonases (PLLs), designing 43 and 34 proteins, respectively. Twenty-one GH10 and seven PLL designs are active, including designs derived from templates with <25% sequence identity. Moreover, four designs are as active as natural enzymes in these families. Atomic accuracy in a high-activity GH10 design is further confirmed by crystallographic analysis. Thus, combinatorial-backbone assembly and design may be used to generate stable, active, and structurally diverse enzymes with altered selectivity or activity.


Assuntos
Técnicas de Química Combinatória , Glicosídeo Hidrolases/química , Hidrolases de Triester Fosfórico/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Cinética , Modelos Moleculares , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica
19.
Curr Opin Chem Biol ; 10(5): 498-508, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16939713

RESUMO

The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.


Assuntos
Enzimas/química , Evolução Molecular , Animais , Catálise , Ativação Enzimática/fisiologia , Enzimas/fisiologia , Humanos , Especificidade por Substrato
20.
Protein Sci ; 26(4): 807-813, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28142198

RESUMO

Allosteric regulation underlies living cells' ability to sense changes in nutrient and signaling-molecule concentrations, but the ability to computationally design allosteric regulation into non-allosteric proteins has been elusive. Allosteric-site design is complicated by the requirement to encode the relative stabilities of active and inactive conformations of the same protein in the presence and absence of both ligand and effector. To address this challenge, we used Rosetta to design the backbone of the flexible heavy-chain complementarity-determining region 3 (HCDR3), and used geometric matching and sequence optimization to place a Zn2+ -coordination site in a fluorescein-binding antibody. We predicted that due to HCDR3's flexibility, the fluorescein-binding pocket would configure properly only upon Zn2+ application. We found that regulation by Zn2+ was reversible and sensitive to the divalent ion's identity, and came at the cost of reduced antibody stability and fluorescein-binding affinity. Fluorescein bound at an order of magnitude higher affinity in the presence of Zn2+ than in its absence, and the increase in fluorescein affinity was due almost entirely to faster fluorescein on-rate, suggesting that Zn2+ preorganized the antibody for fluorescein binding. Mutation analysis demonstrated the extreme sensitivity of Zn2+ regulation on the atomic details in and around the metal-coordination site. The designed antibody could serve to study how allosteric regulation evolved from non-allosteric binding proteins, and suggests a way to designing molecular sensors for environmental and biomedical targets.


Assuntos
Fluoresceína/química , Engenharia de Proteínas , Anticorpos de Cadeia Única , Zinco/química , Sítio Alostérico , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
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