Bernard Badet

Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: Identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties

The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.

Gamma-fluorinated analogues of glutamic acid and glutamine.

Summary. γ-Fluorinated analogues of glutamic acid and glutamine are compounds of biological interest. Syntheses of such compounds are extensively reviewed in this article. 4-Fluoroglutamic acid was prepared as a mixture of racemic diastereomers by Michael reaction, inverse-Michael reaction or by electrophilic / nucleophilic fluorination. Optically enriched 4-fluoroglutamic acids were obtained by several resolution techniques as well as by asymmetric methodologies using the chiral pool. 4-Fluoroglutamine was prepared as a mixture of stereoisomers as well as in racemic erythro and threo forms from the corresponding 4-fluoroglutamic acids using aminolysis and conventional protection and deprotection strategies. Racemic 4,4-difluoroglutamic acid was synthesized by a nitroaldol reaction and its L-enantiomer obtained via three different asymmetric routes. Racemic 4,4-difluoroglutamic acid was converted into the corresponding 4,4-difluoroglutamine using a protection / aminolysis / deprotection sequence while N-Boc-L-4,4-difluoroglutamine was prepared directly from (R)-Garners aldehyde using a Reformatsky reaction as the key step.

Ammonia channeling in bacterial glucosamine-6-phosphate synthase (Glms): molecular dynamics simulations and kinetic studies of protein mutants

Ammonia transfer from the glutamine site to the fructose‐6P site of bacterial glucosamine‐6‐phosphate synthase was studied by molecular dynamics simulations. The studies suggest a key role for Trp74, in the sealing of the hydrophobic channel connecting the two binding sites, as well as for the two Ala602 and Val605 residues, which form a narrow passage whose opening/closing constitutes an essential event in ammonia transfer. Kinetic analyses of the corresponding protein mutants confirmed our predictions. The efficiency of ammonia transfer which was close to zero in the W74A mutant was partially restored by increasing the size of the corresponding side‐chain; the simulations performed on the W74A mutant suggested the formation of a hole in the channel. In the case of A602L and V605L mutants, the efficiency of ammonia transfer decreased to ∼50% of the value of the native protein. None of the mutants were, however, able to use exogenous ammonia as a substrate.

Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: Evidence of histidine requirement for enzymatic activity

Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.

Quantitative Synthesis of L Or D N2-Tertbutoxycarbonyl- 2,3-DIAMINOPROPANOIC ACID FROM PROTECTED L OR D SERINE-β -LACTONE

Abstract The reaction of ammonia on protected L or D serine-β-lactone was reinvestigated. An easy synthesis of N2 -tertbutoxycarbonyl-L-2,3-diaminopropanoic acid (and D enantiomer). an important precursor of several antibiotics is proposed.

A concise synthesis of l-4,4-difluoroglutamine

Abstract l -4,4-Difluoroglutamine 1 of high optical purity was prepared from ( R )-Garners aldehyde 2 using Reformatsky reaction as the key step for introducing the fluorinated side-chain.

Synthesis and evaluation of inhibitors for Escherichia coli glucosamine-6-phosphate synthase

Abstract The design, synthesis and evaluation of potential affinity labels of Escherichia coli glucosamine-6-phosphate synthase (glmS) are described. Among the inhibitors described, 2-amino-3-(( N -halomethyl)amino)propanoates 1a and 2a and 2-amino-3-( N -maleimidyl)propanoate 4a exhibited time-dependent inhibition parameters similar to those previously obtained for N 3 -(4-methoxyfumaroyl)diaminopropanoate 5 , the most efficient synthetic inhibitor of glmS reported to date. From the recently elucidated mechanism of glmS inactivation by 5 , the alkylation of cysteine-1-thiol by 1a, 2a and 4a seems very likely.

Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14N versus 15N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

Inhibition of DNA replication in vitro by pefloxacin

Pefloxacin (a novel quinolone antibiotic) is demonstrated to be a drug inhibiting DNA replication 10‐times more efficiently than oxolinic acid measured either in toluene‐treated E. coli or in an in vitro replication system for oriC plasmids [6]. DNA repair synthesis is not inhibited by the drug.