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1.
Nature ; 586(7827): 145-150, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968273

RESUMO

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

2.
Nat Chem Biol ; 15(7): 669-671, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209348

RESUMO

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and ß-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteína de Transporte de Acila/química , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II/química , Modelos Moleculares , Ligação Proteica
3.
Angew Chem Int Ed Engl ; 58(32): 10888-10892, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31140212

RESUMO

At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo via a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur with other heteroatoms results in significant structural changes, thus suggesting more rigorous selections of isosteric substitutes is needed.


Assuntos
Proteínas de Transporte/química , Ésteres/química , Compostos de Sulfidrila/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Estrutura Molecular
4.
Biochemistry ; 56(40): 5269-5273, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28920687

RESUMO

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.


Assuntos
Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Peptídeo Sintases/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
5.
J Am Chem Soc ; 137(36): 11546-9, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26340431

RESUMO

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems.


Assuntos
Fenóis/química , Pirróis/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos , Conformação Proteica
6.
Chembiochem ; 16(4): 528-547, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25676190

RESUMO

Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in nature, preserving the same handful of chemical reactions across all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug-resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock, or antimicrobial purposes has been met with limited success, due in part to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain will focus first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. Although significant unknowns remain, new understandings of the intricacies of FAS point to future advances in manipulating this complex molecular factory.


Assuntos
Proteína de Transporte de Acila/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteína de Transporte de Acila/química , Animais , Humanos , Modelos Moleculares
7.
Mol Biosyst ; 11(1): 38-59, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25360565

RESUMO

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.


Assuntos
Vias Biossintéticas , Ácidos Graxos/metabolismo , Engenharia Metabólica , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Hidroliases/química , Hidroliases/metabolismo , Engenharia Metabólica/métodos , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo
8.
J Am Chem Soc ; 136(48): 16792-9, 2014 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-25406716

RESUMO

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.


Assuntos
Cetonas/metabolismo , Policetídeo Sintases/química , Cetonas/química , Modelos Moleculares , Conformação Molecular , Policetídeo Sintases/metabolismo
9.
Nature ; 505(7483): 427-31, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24362570

RESUMO

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.


Assuntos
Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Escherichia coli/química , Ácidos Graxos/biossíntese , Sítios de Ligação , Domínio Catalítico , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Histidina/metabolismo , Hidroliases/química , Hidroliases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Mapas de Interação de Proteínas
10.
J Am Chem Soc ; 135(24): 8846-9, 2013 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-23718183

RESUMO

Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein-protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic ß-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.


Assuntos
Proteína de Transporte de Acila/química , Alquinos/química , Reagentes para Ligações Cruzadas/química , Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Sulfonas/química , Proteína de Transporte de Acila/metabolismo , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Modelos Moleculares , Mapeamento de Interação de Proteínas
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