RESUMO
We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.
Assuntos
Acinetobacter/genética , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Deleção de Genes , Mutação , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/fisiologia , Carbono/metabolismo , Mapeamento Cromossômico , Meios de Cultura , Primers do DNA/química , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Modelos Genéticos , Biologia de SistemasRESUMO
The enzyme glucosamine-6P Synthase (Gfat, L-glutamine:D-fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d-fructose-6-phosphate and l-glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N-acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N-acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.
Assuntos
Frutosefosfatos/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/genética , Retroalimentação Fisiológica , Frutosefosfatos/química , Expressão Gênica , Glucosamina/química , Glucosamina/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glutamina/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato N-Acetilglicosamina/químicaRESUMO
Human L-glutamine: D-fructose-6-phosphate amidotransferase (Gfat1), a recognized target in type 2 diabetes complications, was expressed in Sf9 insect cells with an internal His(6)-tag and purified to homogenity. Two different microplate assays that quantify, respectively D-glucosamine-6-phosphate and L-glutamate were used to analyze the enzyme kinetic properties. The recombinant human L-glutamine: D-fructose-6-phosphate amidotransferase isoform 1 exhibits Michaelis parameters K(m)(Fru-6P)=0.98 mM and K(m)(Gln)=0.84 mM which are similar to the values reported for the same enzyme from different sources. The stimulation of hydrolysis of the alternate substrate L-glutamine para-nitroanilide by D-fructose-6P (Fru-6P) afforded a K(d) of 5 microM for Fru-6P.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/biossíntese , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/isolamento & purificação , Histidina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Animais , Células Cultivadas , Dicroísmo Circular , Clonagem Molecular , Ativação Enzimática , Frutosefosfatos/química , Ácido Glutâmico/química , Glutaminase/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Humanos , Hidrólise , Insetos/citologia , Insetos/metabolismo , Cinética , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Results of an in silico screening of a freely accessible database encompassing 50,000 commercial compounds on bacterial glucosamine-6P synthase (Glms) are described. Each product was docked with the GOLD software in a region of 20A surrounding the sugar binding site and ranked according to its score. Among the 14 best-scored molecules, three molecules exhibited good experimental inhibition properties (IC(50)=70 microM) giving a high hit rate (H.R.: 0.23). Interestingly, these molecules are predicted to interact with a protein region that forms a pocket at the interface between the two enzyme monomers, opening the route to dimerization inhibitors.