New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.

Channeling of ammonia in glucosamine-6-phosphate synthase.

Glucosamine-6-phosphate synthase catalyses the first and rate-limiting step in hexosamine metabolism, converting fructose 6-phosphate into glucosamine 6-phosphate in the presence of glutamine. The crystal structure of the Escherichia coli enzyme reveals the domain organisation of the homodimeric molecule. The 18 A hydrophobic channel sequestered from the solvent connects the glutaminase and isomerase active sites, and provides a means of ammonia transfer from glutamine to sugar phosphate. The C-terminal decapeptide sandwiched between the two domains plays a central role in the transfer. Based on the structure, a mechanism of enzyme action and self-regulation is proposed. It involves large domain movements triggered by substrate binding that lead to the formation of the channel.

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance.

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.

The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase.

Glucosamine 6-phosphate synthase converts fructose-6P into glucosamine-6P or glucose-6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40-kDa C-terminal domain, which has already been characterized crystallographically. Now the three-dimensional structures of the complexes with the reaction product glucose-6P and with the transition state analog 2-amino-2-deoxyglucitol-6P have been determined. Glucose-6P binds in a cyclic form whereas 2-amino-2-deoxyglucitol-6P is in an extended conformation. The information on ligand-protein interactions observed in the crystal structures together with the isotope exchange and site-directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine-6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the epsilon-amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.

Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A crystal structure of the isomerase domain.

BACKGROUND: Glucosamine 6-phosphate synthase (GlmS) catalyses the first step in hexosamine metabolism, converting fructose-6P (6 phosphate) into glucosamine-6P using glutamine as a nitrogen source. GlmS is a bienzyme complex consisting of two domains that catalyse glutamine hydrolysis and sugar-phosphate isomerisation, respectively. Knowledge of the three-dimensional structure of GlmS is essential for understanding the general principles of catalysis by ketol isomerases and the mechanism of nitrogen transfer in glutamine amidotransferases. RESULTS: The crystal structure of the isomerase domain of the Escherichia coli GlmS with the reaction product, glucosamine-6P, has been determined at 1.57 A resolution. It is comprised of two topologically identical subdomains, each of which is dominated by a nucleotide-binding motif of a flavodoxin type. The catalytic site is assembled by dimerisation of the protein. CONCLUSIONS: The isomerase active site of GlmS seems to be the result of evolution through gene duplication and subsequent dimerisation. Isomerisation of fructose-6P is likely to involve the formation of a Schiff base with Lys603 of the enzyme, the ring-opening step catalysed by His504, and the proton transfer from C1 to C2 of the substrate effected by Glu488. The highly conserved C-terminal fragment of the chain may play a key role in substrate binding, catalysis and communication with the glutaminase domain. The corresponding sequence pattern DXPXXLAK[SC]VT (in single-letter amino-acid code, where X is any amino acid and letters in brackets indicate that either serine or cysteine may take this position) may be considered as a fingerprint of GlmS.

Modification of kinetoplast DNA minicircle composition in pentamidine-resistant Leishmania.

Pentamidine, an antiprotozoal drug, was shown to have various cellular and molecular targets depending on the organism. In Leishmania, ultrastructural modifications of kinetoplast and mitochondria have been observed but no data is available on cellular and molecular events involved in development of pentamidine-resistance. The absence of modification of minicircle DNA in pentamidine treated L. donovani and L. amazonensis promastigotes suggested that topoisomerase II activity is not a target. This result was confirmed by quantitation of the enzyme by immunodetection. Southern blot experiments indicated that the kDNA network was altered in resistant clones. Molecular cloning and sequence analysis of kDNA minicircles showed transkinetoplastidy hitherto reported only for arsenite- and tunicamycin-resistant Leishmania. Comparison of wild-type and resistant sequences showed only 32-51% homology. The AT-rich regions, known as binding sites, of the drug occurred less frequently in the resistant clones and their locations were different. These minicircle sequence modifications leading to decreased binding sites for the drug might contribute to pentamidine-resistance in Leishmania.

The mechanism of glutamine-dependent amidotransferases.

Glutamine-dependent amidotransferases have been known for more than 30 years. The mechanism by which these enzymes generate ammonia from the glutamine amide nitrogen and transfer it to seven different chemical classes of acceptors has been the subject of intense scrutiny for the last 5 years. The increasing number of biochemical and structural studies dealing with amidotransferases and with mechanistically related enzymes has disclosed the dichotomy of the mechanisms within these enzymes for achieving the glutamine amide bond cleavage. Some of them use a catalytic Cys/His/Glu triad similar to serine protease, whereas the aminoterminal cysteine of the others is believed to play the same function. The transfer of ammonia from the glutamine site to the acceptor site which must operate in a concerted manner has been demonstrated in two cases to involve channelling but is still matter of investigation.

Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the gamma-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k(inact) = 0.60 +/- 0.05 min(-1), Kirr = 1.40 +/- 0.20 mM) by anhydro-1,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of side-chain carboxylates of Glu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne [Bearne, S. L. (1996) J. Biol. Chem. 271, 3052-3057].

Effects of pentamidine on polyamine level and biosynthesis in wild-type, pentamidine-treated, and pentamidine-resistant Leishmania.

Polyamine biosynthesis was studied in wild-type promastigotes of Leishmania donovani and Leishmania amazonensis treated with pentamidine and in the parasites resistant to this drug. Treatment of wild-type clones with low pentamidine concentrations for 24 hr provoked a strong decrease in arginine, ornithine, and putrescine pools, while the level of intracellular spermidine remained unchanged. In these cells, the activity of the enzyme ornithine decarboxylase was found to be decreased. Compared to wild-type cells, resistant clones had a lower level of putrescine, higher pools of arginine and ornithine, and a similar spermidine content. Analysis by Western blot and DFMO-binding showed reduced amount of ornithine decarboxylase. Furthermore, in the resistant cells, the kinetic parameters of the enzyme spermidine synthase were markedly changed, showing increased affinity to putrescine and decreased affinity to pentamidine. Thus, it seems that polyamine biosynthesis pathway is a target of pentamidine in Leishmania and is altered in resistant clones.

Characterization of L-glutamine:D-fructose-6-phosphate amidotransferase from an extreme thermophile Thermus thermophilus HB8.

Glucosamine-6-phosphate synthase from the extremophile Thermus thermophilus (GlmSth) was purified to homogeneity from an Escherichia coli overproducer. The homodimeric enzyme exhibits an optimum activity at 70 degrees C with a half-life of 90 min at 80 degrees C. Dissociation experiments in guanidinium chloride and urea are consistent with the absence of catalytic activity of the monomer. Differential scanning microcalorimetry analysis of GlmSth revealed an irreversible denaturation process with a delta(H)cal = 257 kcal x mol(-1) and Tm = 82.6 degrees C. Antigenic cross-reaction with GlmSth was observed with the E. coli enzyme using monoclonal antibodies (mAbs) specific for linear epitopes of the glutamine binding domain. However, no cross-reactivity was observed with an mAb specific for a native conformation of the E. coli enzyme. The inhibition constants of 6-diazo-5-oxo-L-norleucine and methoxyfumaroyl-L-2,3-diaminopropionic acid, potent glutamine site-directed affinity labels of the E. coli enzyme, were reduced by 2 to 3 orders of magnitude when tested on GlmSth, whereas the properties of 2-amino-2-deoxyglucitol-6P, a potent competitive inhibitor of the fructose-6P site, remained unaffected. These data, combined with its unexpected resistance to limited proteolysis, are consistent with an increase in the structural constraint of the thermophile enzyme vs its mesophilic counterpart.

Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.

BACKGROUND: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS). RESULTS: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 A resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of alpha helices. CONCLUSIONS: The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own alpha-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.

Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase.

In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis. Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity. Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far. The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping. Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach. Neither 505.1 nor 522.2 was directed against linear epitopes. However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor. The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme.

glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene.

A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine-6-P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583.

Acquired resistance to glycopeptides in enterococci is associated with the production of D-Alanine:D-Alanine ligase-related proteins. The VanA protein associated with high-level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D-alanine-D-lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over-production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.

Crystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli.

The glutamine amidohydrolase and fructose 6-phosphate binding domains of glucosamine-6-phosphate synthase from Escherichia coli have been overexpressed, purified and crystallized for X-ray diffraction analysis. The crystals of the glutamine amidohydrolase domain belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 70.4 A, b = 82.5 A, c = 86.1 A, with two molecules in the asymmetric unit, and diffract to 1.9 A resolution. The native Patterson indicated pseudo c-face centering of the unit cell. The fructose 6-phosphate binding domain was crystallized in the hexagonal space group P6(1) or P6(5) with cell dimensions a = b = 63.5 A, c = 334.3 A and with two molecules in the asymmetric unit. Diffraction data to 2.6 A resolution have been collected.

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.