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1.
Nature ; 603(7901): 439-444, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35296845

RESUMO

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.


Assuntos
Guanosina , Nucleotidiltransferases , Adenosina , Animais , Interferons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais
2.
Angew Chem Int Ed Engl ; 63(13): e202316133, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38279624

RESUMO

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.


Assuntos
Indenos , Oxigenases de Função Mista , Oxirredução , Hidroxilação , Biocatálise
3.
ACS Synth Biol ; 10(2): 357-370, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33433999

RESUMO

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.


Assuntos
Mutagênese Sítio-Dirigida/métodos , Mutagênese , Mutação , Oligonucleotídeos/genética , Proteínas/genética , Software , Algoritmos , Códon/genética , Simulação por Computador , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Biblioteca Gênica , Proteínas Mutantes
4.
PLoS Biol ; 4(5): e112, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16594730

RESUMO

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising 3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Engenharia de Proteínas/métodos , Recombinação Genética/genética , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/genética , Estabilidade Enzimática , Temperatura Alta , Lauratos/química , Lauratos/metabolismo , Modelos Logísticos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes , Alinhamento de Sequência
5.
J Mol Biol ; 367(1): 162-73, 2007 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-17254599

RESUMO

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.


Assuntos
Ácido Aspártico/fisiologia , Sequência Conservada/fisiologia , Inteínas/fisiologia , Mycobacterium tuberculosis/enzimologia , Recombinases Rec A/química , Ácido Aspártico/genética , Cristalografia por Raios X , Conformação Proteica , Recombinases Rec A/fisiologia
6.
ACS Synth Biol ; 7(7): 1730-1741, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29782150

RESUMO

Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to create combinations of mutations as part of the initial design was confounded by the relatively large number of predicted mutations that were inactivating (30% and 45% for the sequence-based and structure-based methods, respectively). Despite this, combining several mutations identified within a given method produced variant lysates 7- and 9-fold more active than the wild-type lysate, highlighting the capability of mutations chosen this way to generate large advances in activity in addition to the reductions in screening.


Assuntos
Evolução Molecular Direcionada , Mutagênese/genética , Mutação/genética
7.
J Mol Biol ; 354(4): 916-26, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16288917

RESUMO

Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.


Assuntos
Evolução Molecular Direcionada , Inteínas , Mycobacterium tuberculosis/enzimologia , Recombinases Rec A/química , Recombinases Rec A/genética , Estabilidade Enzimática , Mutação de Sentido Incorreto/fisiologia , Peptídeo Hidrolases/metabolismo , Desnaturação Proteica , Processamento de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Ureia
8.
Biochem J ; 388(Pt 2): 427-33, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15673283

RESUMO

Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.


Assuntos
DNA Bacteriano/química , Poliaminas/química , RNA Bacteriano/química , RNA de Transferência/química , Thermus thermophilus/genética , Desnaturação de Ácido Nucleico , Temperatura , Termodinâmica
9.
Nucleic Acids Res ; 31(16): e91, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12907743

RESUMO

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.


Assuntos
Aminoácidos/genética , Análise Mutacional de DNA/métodos , Mutagênese/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Mutação , Reação em Cadeia da Polimerase , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
10.
J Mol Biol ; 335(4): 1093-104, 2004 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-14698302

RESUMO

To identify peptide units that make up a single-domain protein, we searched for possible combinations of N and C-fragments that exhibit functional complementation, and attempted an exhaustive evaluation of peptide unit boundaries. The tryptophan synthase alpha subunit was used as a model enzyme, which has a single TIM (beta8/alpha8) barrel domain. Libraries comprising randomly digested N and C-fragments were constructed, and clones expressing enzymatic activity were selected by the ability to confer growth of the Escherichia coli trpA mutant on a medium lacking tryptophan. More than 50 clones were obtained, and two cleavable positions were found on the loops after extra-helix 2' and helix 5. Half of the clones harbored N and C-fragments having an overlap between two fragments. The remaining clones harbored one fragment made by the fusion of N and C-fragments with insertional sequence duplication. Mapping the frequency of occurrence of fragment overlap and insertional duplication showed significant peaks at two loops, which coincide with the cleavable sites. These results suggest that the boundaries of unit fragments are located at these positions, and that bisection, fragment overlap and insertion are all possible at the unit boundaries.


Assuntos
Teste de Complementação Genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Triptofano Sintase/química , Triptofano Sintase/metabolismo , Dicroísmo Circular , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Deleção de Sequência/genética , Relação Estrutura-Atividade , Triptofano/biossíntese , Triptofano/metabolismo , Triptofano Sintase/genética
11.
J Mol Biol ; 330(2): 287-96, 2003 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-12823968

RESUMO

We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined beta-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genes created, and screened the library of 2(8) (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.


Assuntos
Evolução Molecular Direcionada/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Algoritmos , Sequência de Bases , Sítios de Ligação/genética , Quimera/genética , DNA Recombinante/genética , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Plasmídeos/genética , Recombinação Genética , beta-Lactamases/genética
12.
J Biomol Screen ; 20(7): 869-75, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26024945

RESUMO

For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Imunofluorescência , Corantes Fluorescentes , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Imunoconjugados/farmacologia , Transporte Proteico , Proteólise
13.
J Mol Biol ; 393(5): 1106-17, 2009 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-19744499

RESUMO

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a "ripple effect", whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.


Assuntos
Biocatálise , Inteínas , Mycobacterium tuberculosis/química , Biblioteca de Peptídeos , Sequência de Aminoácidos , Quitina , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Mutantes/química , Mutação/genética , Fenótipo , Processamento de Proteína , Estrutura Secundária de Proteína , Resinas Sintéticas
14.
Curr Protoc Protein Sci ; Chapter 26: 26.2.1-26.2.17, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18429308

RESUMO

Recombination of distantly related and nonrelated genes is difficult using traditional PCR-based techniques, and truncation-based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments.


Assuntos
Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Sequência de Bases , Primers do DNA , Plasmídeos , Reação em Cadeia da Polimerase
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