ABSTRACT
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.
Subject(s)
Fructosephosphates/metabolism , Glucosamine/analogs & derivatives , Glucose-6-Phosphate/analogs & derivatives , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/chemistry , Glutamine/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , Catalytic Domain , Escherichia coli/enzymology , Escherichia coli/genetics , Feedback, Physiological , Fructosephosphates/chemistry , Gene Expression , Glucosamine/chemistry , Glucosamine/metabolism , Glucose-6-Phosphate/chemistry , Glucose-6-Phosphate/metabolism , Glutamine/chemistry , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
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.
Subject(s)
Acinetobacter/genetics , Bacterial Proteins/genetics , Escherichia coli/metabolism , Gene Deletion , Mutation , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/physiology , Carbon/metabolism , Chromosome Mapping , Culture Media , DNA Primers/chemistry , Gene Expression Regulation, Bacterial , Models, Biological , Models, Genetic , Systems BiologyABSTRACT
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.
Subject(s)
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/biosynthesis , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/isolation & purification , Histidine/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Animals , Cells, Cultured , Circular Dichroism , Cloning, Molecular , Enzyme Activation , Fructosephosphates/chemistry , Glutamic Acid/chemistry , Glutaminase/chemistry , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/chemistry , Humans , Hydrolysis , Insecta/cytology , Insecta/metabolism , Kinetics , Protein Structure, Tertiary , Recombinant Proteins/chemistryABSTRACT
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.
