Henryk Chmara

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.

Synthetic derivatives of N3-fumaroyl-l-2,3-diaminopropanoic acid inactivate glucosamine synthetase from Candida albicans

Synthetic derivatives of N3-fumaroyl-L-2,3-diaminopropanoic acid constitute the novel group of glutamine analogs. They are powerful, competitive inhibitors of the glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19) from Candida albicans with respect to glutamine and uncompetitive with respect to D-fructose 6-phosphate. Some of the compounds tested irreversibly inactivate glucosamine synthetase with Kinact values of 10(-4) to 10(-6) M. The addition of glutamine protects enzyme from the inactivation, while the absence of D-fructose 6-phosphate lowers the rate of inactivation. An ordered, sequential mechanism is suggested for binding of the inhibitors to the glutamine-binding site. A number of tested compounds act as active-site-directed, irreversible inhibitors. It is suggested that derivatives of N3-fumaroyl-L-2,3-diaminopropanoic acid should be classified as mechanism-based enzyme inactivators. Structural requirements for an effective inactivator containing N3-fumaroyl-L-2,3-diaminopropanoic acid moiety are discussed.

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT 2 SL 1027 by Nβ-fumarylcarboxyamido-L-2,3-diaminopropionic acid

N beta- fumarylcarboxyamido -L-2,3-diaminopropionic acid ( FCDP ), a novel glutamine analog, inhibits the reaction of glucosamine-6-phosphate synthetase (EC 5.3.1.19) from Salmonella typhimurium LT 2 by irreversible inactivation of the enzyme. The kinetic data on enzyme inhibition and inactivation are presented. It is suggested that the enzyme inactivation occurs according to a sequential mechanism.

Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase

Several dipeptides, containing the N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis. FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases. Cleavage rates [0.1-5.6 micromol min-1 (mg protein)-1] were about two orders of magnitude lower than transport rates [40-200 micromol min-1 (mg dry wt)-1]. The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan. Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect. Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis. Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.

Bacteriolytic effect of teicoplanin

The glycopeptide antibiotic teicoplanin belongs to the same group as vancomycin and ristocetin and is a valuable tool for studying the autolytic system of sensitive Gram-positive bacteria. Teicoplanin, at a concentration of 1 microgram ml-1, caused rapid lysis of exponential phase cells of Streptococcus faecalis. Bacillus spp. were most sensitive to the antibiotic; effective lysis occurred at 0.1 microgram teicoplanin ml-1. The bacteriolytic effect depended on the antibiotic concentration, the growth phase and growth rate of the target organism. Antibiotic added to overnight cultures did not cause lysis. Mg2+ (50 mM) was unable to prevent lysis. Mutants with decreased autolytic activity were more resistant to teicoplanin and lysed more slowly than the wild-type. Growth of bacteria in slightly acidic medium protected the cells against the lytic effect of teicoplanin typically observed at pH 7 or 8. This pH-dependent antibiotic tolerance was demonstrated with both bacilli and streptococci. Bacterial lysis was prevented by the presence of Ac-L-Lys(Ac)-D-Ala-D-Ala and normal growth was observed when this peptide was added simultaneously with teicoplanin. Bacteria pretreated with teicoplanin, washed and transferred to fresh medium or buffers behaved as if the antibiotic was still present; in neutral or slightly alkaline conditions strong lysis occurred, whereas in acidic buffer only bacteriostasis was observed. In contrast to vancomycin, teicoplanin induced some lysis of bacteria in hypertonic media, presumably by affecting the integrity of the cell membrane.