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1.
Nat Commun ; 12(1): 6933, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34836937

RESUMO

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.


Assuntos
Proteínas de Bactérias/ultraestrutura , Complexo Piruvato Desidrogenase/ultraestrutura , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Chaetomium/enzimologia , Coenzima A/metabolismo , Coenzima A/ultraestrutura , Microscopia Crioeletrônica , Ensaios Enzimáticos , Redes e Vias Metabólicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo Piruvato Desidrogenase/metabolismo
2.
Biochim Biophys Acta Proteins Proteom ; 1869(2): 140566, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33271445

RESUMO

Phosphopantetheine Adenylyltransferase (PPAT) is an enzyme that catalyzes the penultimate step in the biosynthesis of Coenzyme A (CoA), which is the active and physiologically functional form of dietary Vitamin B5. CoA serves as a cofactor for numerous metabolic reactions which makes it essential for cellular survival. This enzyme is also subject to feedback inhibition by CoA to maintain its cellular concentration. The steps of the CoA biosynthesis pathway remain conserved from prokaryotes to eukaryotes, with humans and pathogenic micro-organisms showing significant diversity on a sequence, structure and mechanistic level. This suggests that the development of selective inhibitors of microbial CoA biosynthesis should be possible using these enzymes as targets for drug development. Bacterial PPAT shows significant mechanistic difference from its human counterpart CoA synthase, which is a dual protein carrying the activity of both PPAT and next step in the pathway catalyzed by the enzyme Dephospho CoA kinase (DPCK). This review covers the detailed description of the mechanistic, structural and functional aspects of this enzyme. Also, all the attempts to design high efficiency inhibitors of this enzyme using the approach of structure based drug design have been discussed in detail. This comprehensive structural and functional discussion of PPAT will help in further exploiting it as a drug target.


Assuntos
Coenzima A/química , Farmacorresistência Bacteriana/genética , Nucleotidiltransferases/química , Coenzima A/ultraestrutura , Cristalografia por Raios X , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/ultraestrutura , Conformação Proteica , Relação Estrutura-Atividade
3.
Sci Rep ; 8(1): 10692, 2018 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013155

RESUMO

Metallosphaera sedula is a thermoacidophilic autotrophic archaeon known to utilize the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) as carbon fixation pathway. 3-Hydroxypropionyl-CoA dehydratase (3HPCD) is an enzyme involved in the 3-HP/4-HB cycle by converting 3-hydroxypropionyl-CoA to acryloyl-CoA. To elucidate the molecular mechanism of 3HPCD from M. sedula (Ms3HPCD), we determined its crystal structure in complex with Coenzyme A (CoA). Ms3HPCD showed an overall structure and the CoA-binding mode similar to other enoyl-CoA hydratase (ECH) family enzymes. However, compared with the other ECHs, Ms3HPCD has a tightly formed α3 helix near the active site, and bulky aromatic residues are located at the enoyl-group binding site, resulting in the enzyme having an optimal substrate binding site for accepting short-chain 3-hydroxyacyl-CoA as a substrate. Moreover, based on the phylogenetic tree analysis, we propose that the 3HPCD homologues from the phylum Crenarchaeota have an enoyl-group binding pocket similar to that of bacterial short-chain ECHs.


Assuntos
Proteínas Arqueais/ultraestrutura , Ciclo do Carbono , Hidroliases/ultraestrutura , Sulfolobaceae/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Coenzima A/metabolismo , Coenzima A/ultraestrutura , Cristalografia por Raios X , Hidroliases/genética , Hidroliases/metabolismo , Hidroxibutiratos/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Simulação de Acoplamento Molecular , Filogenia , Estrutura Quaternária de Proteína , Especificidade por Substrato , Sulfolobaceae/genética
4.
Nanoscale ; 4(2): 659-66, 2012 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-22159287

RESUMO

The development of a novel method for functionalizing nanopatterned surfaces with catalytically active proteins is reported. This method involves using dip-pen nanolithography (DPN) and polymer pen lithography (PPL) to generate nanoscale patterns of coenzyme A, followed by a phosphopantetheinyl transferase-mediated coupling between coenzyme A and proteins fused to the ybbR-tag. By exploiting the ability to generate protein features over large areas afforded by DPN and PPL, it was now possible to measure protein activity directly on these surfaces. It was found that proteins immobilized on the nanoscale features not only display higher activity per area with decreasing feature size, but are also robust and can be used for repeated catalytic cycles. The immobilization method is applicable to a variety of proteins and gives rise to superior activity compared to proteins attached in random orientations on the surface.


Assuntos
Materiais Revestidos Biocompatíveis/síntese química , Coenzima A/química , Coenzima A/ultraestrutura , Periféricos de Computador , Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Ativação Enzimática , Enzimas Imobilizadas , Substâncias Macromoleculares/química , Teste de Materiais , Microfluídica/métodos , Conformação Molecular , Tamanho da Partícula , Propriedades de Superfície
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