RESUMO
Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.
Assuntos
Sistema Livre de Células/metabolismo , Glucosamina 6-Fosfato N-Acetiltransferase/antagonistas & inibidores , Glucosamina 6-Fosfato N-Acetiltransferase/biossíntese , Sequência de Aminoácidos , Sítios de Ligação , Biotecnologia/métodos , Glucosamina 6-Fosfato N-Acetiltransferase/química , Glucosamina 6-Fosfato N-Acetiltransferase/genética , Glucose-6-Fosfato/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de SequênciaRESUMO
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.