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S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC(50) values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.

7,8-Diaminopelargonic acid aminotransferase (DAPA AT), a potential drug target in Mycobacterium tuberculosis, transforms 8-amino-7-oxononanoic acid (KAPA) into DAPA. We have designed an analytical method to measure the enantiomeric excess of KAPA, based on the derivatization of its amine function, by ortho-phtalaldehyde and N-acetyl-l-cysteine, followed by high pressure liquid chromatography separation. Using this methodology and enantiopure samples of KAPA it appeared that racemization of KAPA occurs rapidly (half-lives from 1 to 8 h) not only in 4 M HCl but more importantly in the usual pH range, from 7 to 9. Furthermore, we showed that racemic KAPA, and not enantiopure KAPA, was used in all previous studies. The only valid enantioselective synthesis of KAPA is that reported by Lucet et al. (1996) Tetrahedron: Asymmetry 7, 985-988. KAPA is produced as a pure (S)-enantiomer by KAPA synthase and by microbial production and DAPA AT only uses (S)-KAPA as substrate. However, (R)-KAPA is an inhibitor of this enzyme. It binds to the pyridoxal 5'-phosphate form (K(i1) = 5.9 +/- 0.2 microM) and to the pyridoxamine 5'-phosphate form (K(i2) = 1.7 +/- 0.2 microM) of M. tuberculosis DAPA AT. Molecular modeling showed that (R)-KAPA forms specific hydrogen bonds with T309 and the phosphate group of the cofactor of DAPA AT. Desmethyl-KAPA (8-amino-7-oxooctanoic acid), an achiral analog of KAPA, is also a potent inhibitor of M. tuberculosis DAPA AT. This molecule binds to the enzyme in a similar way than (R)-KAPA with the following constants: K(i1) = 4.2 +/- 0.2 microM, and K(i2) = 0.9 +/- 0.2 microM. These findings pave the way to the design of new antimycobacterial drugs.

Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S-adenosyl-l-methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5'-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pK(a) of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: K(mAdoMet) = 0.78 +/- 0.20 mm, K(mKAPA) = 3.8 +/- 1.0 microm, k(cat) = 1.0 +/- 0.2 min(-1), K(iKAPA) = 14 +/- 2 microm. Amiclenomycin and a new analogue, 4-(4c-aminocyclohexa-2,5-dien-1r-yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: K(i) = 12 +/- 2 microm, k(inact) = 0.35 +/- 0.05 min(-1), and K(i) = 20 +/- 2 microm, k(inact) = 0.56 +/- 0.05 min(-1), for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 microg.mL(-1)) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo.

Abstract: The four last steps of biotin biosynthesis, starting from pimeloyl-CoA, are conserved among all the biotin-producing microorganisms. Two enzymes of this pathway, the 8-amino-7-oxononanoate synthase (AONS) and the 7,8-diaminopelargonic acid aminotransferase (DAPA AT) are dependent on pyridoxal-5′-phosphate (PLP). This review summarizes our current understanding of the structure, reaction mechanism and inhibition on these two interesting enzymes. Mechanistic studies as well as the determination of the crystal structure of AONS have revealed a complex mechanism involving an acylation with inversion of configuration and a decarboxylation with retention of configuration. This reaction mechanism is shared by the homologous 5-aminolevulinate synthase and serine palmitoyltransferase. While the reaction catalyzed by DAPA AT is a classical PLP-dependent transamination, the inactivation of this enzyme by amiclenomycin, a natural antibiotic that is active against Mycobacterium tuberculosis, involves the irreversible formation of an adduct between PLP and amiclenomycin. Mechanistic and structural studies allowed the complete description of this unique inactivation mechanism. Several potent inhibitors of these two PLP-dependent enzymes have been prepared and might be useful as starting points for the design of herbicides or antibiotics. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology. [Copyright &y& Elsevier]

Cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acid using S-adenosyl-L-methionine as the methylene donor. The crystal structure of three cyclopropane synthases from Mycobacterium tuberculosis showed a bicarbonate ion bound in the active site that was proposed to act as a general base in the reaction mechanism [Huang, C., Smith, V., Glickman, M. S., Jacobs, W. R., and Sacchettini, J. C. (2002) J. Biol. Chem. 277, 11559-11569]. Because the in vitro activity of M. tuberculosis cyclopropane synthases has not yet been reported and because the ligands of the bicarbonate ion are all strictly conserved in cyclopropane synthases, we used the closely related Escherichia coli cyclopropane fatty acid synthase for this study. The putative ligands that share a hydrogen bond with the bicarbonate through their side chains were mutated. H266A, Y3 17F, E239A, and E239Q mutants were thus constructed and purified, and their catalytic efficiencies were 5.3, 0.7, 0.2, and <0.02%, respectively. C 139 that is bound to the bicarbonate by its NH amide had already been mutated to serine in a previous work, and this mutant retains 31 % of the activity of the wild-type enzyme. Kinetic analyses and binding studies using spectrofluorimetry showed that these mutations affected the catalytic constant rather than the binding of the substrates. While addition of free bicarbonate had almost no effect on the wild-type enzyme activity, all mutants, with the exception of E239A and E239Q, were rescued by the addition of free bicarbonate. The catalytic efficiencies of the rescued mutants were 85, 16, and 14% for C139S, H266A, and Y317F, respectively. This effect was specific to bicarbonate. The kinetic parameters of the rescued mutants were determined, and it is shown that the rescuing effect is due to an increase in kcat. These data are interpreted by assuming that the E. coli cyclopropane fatty acid synthase specifically binds a bicarbonate ion that is involved in catalysis, as proposed for the M. tuberculosis enzymes, and that mutation of the bicarbonate ligands decreases the affinity for that ion. However, because the E239Q mutation could not be rescued, we propose that E239 forms a catalytic dyad with the bicarbonate to perform the proton abstraction necessary in the chemical pathway to the cyclopropane ring. [ABSTRACT FROM AUTHOR]

Abstract: 7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20nM to 50μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. [Copyright &y& Elsevier]

Abstract: Bacterial cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acids by transferring a methylene group from S-adenosyl-l-methionine (AdoMet) to the double bond of the lipids. Mycobacterium tuberculosis cyclopropane synthases have been shown to be implicated in pathogenicity, and therefore constitute attractive targets for the development of new drugs against tuberculosis. However, no in vitro assay for these cyclopropane synthases has yet been described. The homologous E. coli enzyme, cyclopropane fatty acid synthase, is thus a valuable model for inhibitor screening. Here, we report the adaptation to the E. coli CFAS of a previously reported enzyme-coupled colorimetric assay based on the quantification, using Ellman''s reagent, of homocysteine produced from S-adenosyl-l-homocysteine, a product of the reaction, in the presence of AdoHcy nucleosidase and S-ribosylhomocysteinase. Using this assay we measured the kinetic parameters for CFAS: K m (AdoMet)=80 μM, k cat =4 min−1. We adapted this assay to microtiter plates and tested 15 potential inhibitors of CFAS. Among them, two new inhibitors, a lipid analog and a thioether analog of AdoHcy, showed IC50 of 4 μM and 11 μM, respectively. This new assay will thus be useful for high-throughput screening of compound libraries for discovering novel antituberculous drug candidates. [Copyright &y& Elsevier]