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1.
Science ; 369(6502): 440-445, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703877

RESUMO

The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.


Assuntos
Corismato Mutase , Evolução Molecular , Modelos Genéticos , Modelos Estatísticos , Sequência de Aminoácidos , Corismato Mutase/química , Corismato Mutase/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética
2.
J Biol Chem ; 295(51): 17514-17534, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33453995

RESUMO

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m-1s-1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.


Assuntos
Proteínas de Bactérias/metabolismo , Corismato Mutase/metabolismo , Mycobacterium tuberculosis/enzimologia , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Corismato Mutase/química , Corismato Mutase/genética , Cristalografia por Raios X , Evolução Molecular Direcionada , Cinética , Simulação de Dinâmica Molecular , Mutagênese , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Ácido Chiquímico/metabolismo , Temperatura de Transição
3.
Biochemistry ; 57(5): 557-573, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29178787

RESUMO

Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM-CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Corismato Mutase/metabolismo , Corynebacterium glutamicum/enzimologia , Triptofano/metabolismo , Regulação Alostérica , Aminoácidos Aromáticos/metabolismo , Corismato Mutase/química , Corynebacterium glutamicum/química , Corynebacterium glutamicum/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Fenilalanina/metabolismo , Conformação Proteica , Multimerização Proteica , Ácido Chiquímico/metabolismo , Tirosina/metabolismo
4.
J Mol Biol ; 428(6): 1237-1255, 2016 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-26776476

RESUMO

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Regulação Alostérica , Corismato Mutase/química , Corismato Mutase/metabolismo , Mycobacterium tuberculosis/enzimologia , Ácido Chiquímico/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Difusão Dinâmica da Luz , Redes e Vias Metabólicas , Ligação Proteica , Espalhamento a Baixo Ângulo
5.
Proc Natl Acad Sci U S A ; 111(49): 17516-21, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422475

RESUMO

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.


Assuntos
Bacillus subtilis/enzimologia , Corismato Mutase/química , Catálise , Domínio Catalítico , Ácido Corísmico/química , Citrulina/química , Cristalização , Cristalografia por Raios X , Ácidos Cicloexanocarboxílicos/química , Cicloexenos/química , Elétrons , Escherichia coli/metabolismo , Cinética , Ligantes , Modelos Moleculares , Oxigênio/química , Conformação Proteica , Dobramento de Proteína , Eletricidade Estática
6.
PLoS One ; 9(12): e116234, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25551646

RESUMO

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.


Assuntos
Corismato Mutase/metabolismo , Evolução Molecular Direcionada/métodos , Mycobacterium tuberculosis/metabolismo , Mapeamento de Interação de Proteínas/métodos , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Calibragem , Corismato Mutase/genética , Biblioteca Gênica , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Distribuição Aleatória
7.
J Struct Biol ; 185(2): 168-77, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23537847

RESUMO

Computational methods have been recently applied to the design of protein-protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (KD=100µM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >10(2)-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein-protein interfaces.


Assuntos
Proteínas Recombinantes de Fusão/química , Quinases Ativadas por p21/química , Sequência de Aminoácidos , Simulação por Computador , Evolução Molecular Direcionada , Citometria de Fluxo , Polarização de Fluorescência , Humanos , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae
8.
Nat Chem Biol ; 9(8): 494-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23748672

RESUMO

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.


Assuntos
Aldeído Liases/química , Aldeído Liases/metabolismo , Biologia Computacional , Evolução Molecular Direcionada , Biocatálise , Domínio Catalítico , Simulação por Computador , Modelos Moleculares , Estrutura Molecular
9.
PLoS Genet ; 9(1): e1003187, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23300488

RESUMO

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.


Assuntos
Aminoácidos , Evolução Molecular Direcionada , Código Genético , Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Corismato Mutase/química , Corismato Mutase/genética , Methanococcales/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade
10.
Chem Biodivers ; 9(11): 2507-27, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23161632

RESUMO

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non-compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard therapies in use for over 50 years and only few new candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development of new medication is structure-guided design, combined with virtual screening or docking studies. Here, we report the results of a drug-design project, which we based on a publication that claimed the structure-guided discovery of several promising and highly active inhibitors targeting the secreted chorismate mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives, show any inhibitory effects against *MtCM.


Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Corismato Mutase/antagonistas & inibidores , Desenho de Fármacos , Mycobacterium tuberculosis/enzimologia , Corismato Mutase/metabolismo , Humanos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
11.
Protein Sci ; 21(5): 717-26, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22407837

RESUMO

Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water-mediated stabilization of the carbinolamine hydroxyl and ß-hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.


Assuntos
Aldeído Liases/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Domínio Catalítico , Evolução Molecular Direcionada/métodos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Biochemistry ; 50(40): 8594-602, 2011 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-21916414

RESUMO

Degradation tags are short peptide sequences that target proteins for destruction by housekeeping proteases. We previously utilized the C-terminal SsrA tag in directed evolution experiments to decrease the intracellular lifetime of a growth-limiting enzyme and thereby facilitate selection of highly active variants. In this study, we examine the N-terminal RepA tag as an alternative degradation signal for laboratory evolution. Although RepA proved to be less effective than SsrA at lowering protein concentrations in the cell, its N-terminal location dramatically reduced the occurrence of truncation and frameshift artifacts in selection experiments. We exploited this improvement to evolve a topologically redesigned chorismate mutase that is intrinsically disordered but already highly active for the conversion of chorismate to prephenate. After three rounds of mutagenesis and high-stringency selection, a robust and more nativelike variant was obtained that exhibited a catalytic efficiency (k(cat)/K(M) = 84000 M(-1) s(-1)) comparable to that of a natural dimeric chorismate mutase. Because of concomitant increases in catalyst yield, the level of intracellular prephenate production increased approximately 30-fold overall over the course of evolution.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Corismato Mutase/química , Corismato Mutase/metabolismo , Evolução Molecular Direcionada/métodos , Methanococcales/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Arqueais/genética , Catálise , Corismato Mutase/genética , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexenos/metabolismo , Cinética , Methanococcales/química , Methanococcales/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas
14.
J Mol Biol ; 399(4): 541-6, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20433850

RESUMO

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function.


Assuntos
Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Corismato Mutase/química , Corismato Mutase/genética , Corismato Mutase/metabolismo , Sequência Consenso , DNA Bacteriano/genética , Estabilidade Enzimática , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Filogenia , Conformação Proteica , Estabilidade Proteica , Proteínas/metabolismo , Alinhamento de Sequência , Termodinâmica
15.
Protein Sci ; 19(5): 1000-10, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20306491

RESUMO

Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.


Assuntos
Corismato Mutase/química , Corismato Mutase/metabolismo , Evolução Molecular Direcionada/métodos , Engenharia de Proteínas/métodos , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Corismato Mutase/genética , Corismato Mutase/isolamento & purificação , Técnicas de Química Combinatória/métodos , Escherichia coli/genética , Zíper de Leucina , Mathanococcus/enzimologia , Mathanococcus/genética , Dados de Sequência Molecular , Mutagênese , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
16.
Biochim Biophys Acta ; 1804(4): 752-4, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19948253

RESUMO

The (13)C isotope effect for the conversion of prephenate to phenylpyruvate by the enzyme prephenate dehydratase from Methanocaldococcus jannaschii is 1.0334+/-0.0006. The size of this isotope effect suggests that the reaction is concerted. From the X-ray structure of a related enzyme, it appears that the only residue capable of acting as the general acid needed for removal of the hydroxyl group is threonine-172, which is contained in a conserved TRF motif. The more favorable entropy of activation for the enzyme-catalyzed process (25 eu larger than for the acid-catalyzed reaction) has been explained by a preorganized microenvironment that obviates the need for extensive solvent reorganization. This is consistent with forced planarity of the ring and side chain, which would place the leaving carboxyl and hydroxyl out of plane. Such distortion of the substrate may be a major contributor to catalysis.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Methanococcales/enzimologia , Prefenato Desidratase/química , Prefenato Desidratase/metabolismo , Proteínas Arqueais/genética , Isótopos de Carbono , Catálise , Domínio Catalítico , Entropia , Ativação Enzimática , Cinética , Methanococcales/genética , Prefenato Desidratase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/química
17.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 65(Pt 10): 1048-52, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19851019

RESUMO

Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM-MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM-MtDS complex diffracted to 1.6 and 2.1 A resolution, respectively.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/química , Corismato Mutase/química , Mycobacterium tuberculosis/enzimologia , Cristalização , Cristalografia por Raios X , Complexos Multienzimáticos/química
18.
EMBO J ; 28(14): 2128-42, 2009 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-19556970

RESUMO

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active-site residues. However, its catalytic efficiency increases >100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme. The 2.35 A crystal structure of the MtCM-MtDS complex bound to a transition-state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active-site residues on binding to MtDS. The mutagenesis of the C-terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate-mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non-covalent enzyme complexes comprising an AroQ(delta) subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/química , Corismato Mutase/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Corismato Mutase/genética , Corismato Mutase/metabolismo , Clonagem Molecular , Corynebacterium glutamicum/enzimologia , Cristalografia por Raios X , Ativação Enzimática , Malatos/química , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Fenilalanina/metabolismo , Alinhamento de Sequência , Ácido Chiquímico/metabolismo , Tirosina/metabolismo
19.
Mol Plant Pathol ; 10(2): 189-200, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19236568

RESUMO

In this article, we present the cloning of Hscm1, a gene for chorismate mutase (CM) from the beet cyst nematode Heterodera schachtii. CM is a key branch-point enzyme of the shikimate pathway, and secondary metabolites that arise from this pathway control developmental programmes and defence responses of the plant. By manipulating the plant's endogenous shikimate pathway, the nematode can influence the plant physiology for its own benefit. Hscm1 is a member of the CM gene family and is expressed during the pre-parasitic and parasitic stages of the nematode's life cycle. In situ mRNA hybridization reveals an expression pattern specific to the subventral and dorsal pharyngeal glands. The predicted protein has a signal peptide for secretion in addition to two domains. The N-terminal domain of the mature protein, which is only found in cyst nematodes, contains six conserved cysteine residues, which may reflect the importance of disulphide bond formation for protein stabilization. The C-terminal domain holds a single catalytic site and has similarity to secreted CMs of pathogenic bacteria, classifying HsCM1 as an AroQ(gamma) enzyme. The presumed catalytic residues are discussed in detail, and genetic complementation experiments indicate that the C-terminal domain is essential for enzyme activity. Finally, we show how the modular design of the protein is mirrored in the genomic sequence by the intron/exon organization, suggesting exon shuffling as a mechanism for the evolutionary assembly of this protein.


Assuntos
Corismato Mutase/química , Corismato Mutase/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Filogenia , Plantas/parasitologia , Tylenchoidea/enzimologia , Sequência de Aminoácidos , Animais , Southern Blotting , Corismato Mutase/genética , Células Eucarióticas/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Teste de Complementação Genética , Dados de Sequência Molecular , Parasitos/enzimologia , Células Procarióticas/enzimologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Tylenchoidea/genética
20.
J Am Chem Soc ; 130(46): 15361-73, 2008 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-18939839

RESUMO

Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mechanical transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites observed by X-ray crystallography was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mechanically optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theoretical ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quantitatively defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.


Assuntos
Domínio Catalítico , Esterases/química , Esterases/metabolismo , Biocatálise , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína
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