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2.
Biochemistry ; 56(37): 4951-4961, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28816437

RESUMO

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Aspartato Aminotransferases/antagonistas & inibidores , Cicloleucina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Fosfato de Piridoxal/metabolismo , 4-Aminobutirato Transaminase/química , 4-Aminobutirato Transaminase/metabolismo , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cicloleucina/química , Cicloleucina/metabolismo , Cicloleucina/farmacologia , Bases de Dados de Compostos Químicos , Bases de Dados de Proteínas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ligantes , Conformação Molecular , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Conformação Proteica , Fosfato de Piridoxal/química , Piridoxamina/química , Piridoxamina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 114(15): 3891-3896, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28348215

RESUMO

The Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosfato de Piridoxal/metabolismo , Ácido gama-Aminobutírico/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Óperon , Ácidos Pentanoicos/metabolismo , Ácidos Pentanoicos/farmacologia , Regiões Promotoras Genéticas , Domínios Proteicos , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética , Bases de Schiff , Transcrição Gênica , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/genética
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