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Cyanobacteria contain pigments, which generate auto-fluorescence that interferes with fluorescence in situ hybridization (FISH) imaging of cyanobacteria. We describe simple chemical treatments using CuSO 4 or H 2 O 2 that significantly reduce the auto-fluorescence of Microcystis strains. These protocols were successfully applied in FISH experiments using 16S rRNA specific probes and filamentous cyanobacteria. [ABSTRACT FROM AUTHOR]

Abstract: Cyanobacteria produce a wide range of secondary metabolites that are very diverse in chemical structure. These metabolites show also very diverse biological activities, including cytotoxicity, neurotoxicity, dermatotoxicity, and inhibition of proteases. The cyanotoxins that are harmful to animals, including humans, are essentially produced by freshwater cyanobacteria, while marine and terrestrial cyanobacteria produce some metabolites that are promising new drugs. Between 2001 and 2010, many biosyntheses of cyanobacterial secondary metabolites have been deciphered at the genetic and biochemical level. Thanks to the advent of genomic data on cyanobacterial genomes and to new powerful bioinformatic tools, about 30 clusters of genes responsible for the production of cyanobacterial secondary metabolites have been identified. The biosyntheses have also been studied in vitro, in certain cases. For instance, among the cyanotoxins, the microcystin, the cylindrospermopsin, the saxitoxin and the anatoxin-a biosyntheses have been elucidated. Almost all cyanobacterial secondary metabolites are the products of polyketide synthases, nonribosomal peptide synthases or hybrid thereof. However, ribosomal peptides are also produced by cyanobacteria, like the cyanobactins and recent genome mining data suggest that these metabolites are more represented than first thought in cyanobacteria. This review gives an overview of the connections between cyanobacterial secondary metabolites and their biosynthetic genes, with emphasis on the most significant cases like the cyanotoxins, sunscreens, alkanes and terpenes. [Copyright &y& Elsevier]

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]

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 p Ka 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: KmAdoMet = 0.78 ± 0.20 mm, KmKAPA = 3.8 ± 1.0 µm, kcat = 1.0 ± 0.2 min−1, KiKAPA = 14 ± 2 µm. Amiclenomycin and a new analogue, 4-(4 c-aminocyclohexa-2,5-dien-1 r-yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: Ki = 12 ± 2 µm, kinact = 0.35 ± 0.05 min−1, and Ki = 20 ± 2 µm, kinact = 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 µg·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 FROM AUTHOR]

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]

8‐Amino‐7‐oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro‐organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8‐amino‐7‐oxopelargonate synthase using six different compounds. d‐Alanine, the enantiomer of the substrate of this pyridoxal‐5′‐phosphate‐dependent enzyme was found to be a competitive inhibitor with respect to l‐alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l‐alanine was interpreted as the consequence of the inversion‐retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d‐alanine and pyridoxal‐5′‐phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d‐alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d‐alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl‐CoA, the second substrate. This result strongly suggests that the binding of pimeloyl‐CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d‐alanine and to the putative reaction intermediate since they both have the same configuration. (±)‐8‐Amino‐7‐oxo‐8‐phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 µm. This compound behaved as a reversible slow‐binding inhibitor, competitive with respect to l‐alanine. Kinetic investigation showed that... [ABSTRACT FROM AUTHOR]

Studies the biosynthesis of ascarylose. Catalytic efficiency of the system used in the experiment; Use of hybrid system in scoring interactions between proteins coexpressed in yeast.

Studies the inactivation of the enzyme CDP-6-deoxy-L-threo-D4-hexulose 3-dehydrase (E1) from Yersinia pseudotuberculosis by diethyl pyrocarbonate (DEP). Formation of 3,6-dideoxyhexose; Cause of DEP's inactivation of E1; Protection against DEP inactivation; Invariant residues conserved in the E1 sequence; Replacement of lysine by a histidine residue (H220).

The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7-oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over-expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine-pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using ¹H NMR. Spectra of the 8-amino-7-oxopelargonate formed using either L-[2-²H]alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino-7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L-alanine with the solvent protons. Similarly, 8-amino-7-oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7-oxopelargonate. In addition to these exchange reactions, 8-amino-7-oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino-7-oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch. = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans. = 0.11 h-1 for the transmination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0). The magnitudes of the KIE are consisted with a partially rate-limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step. Taken together, all these results show that 8-amino-7-oxopelargonate synthase utilizes mechanism (a). 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of α-oxoamines. Based on the fact that all these α-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism. [ABSTRACT FROM AUTHOR]

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/ mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 µM and a kcat/Km of 1.8 × 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyI-CoA above 10 µM. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase. [ABSTRACT FROM AUTHOR]

Abstract: Cyclopropanation of unsaturated lipids is an intriguing enzymatic reaction and a potential therapeutic target in Mycobacterium tuberculosis. Cyclopropane fatty acid synthase from Escherichia coli is the only in vitro model available to date for mechanistic and inhibition studies. While the overall reaction mechanism of this enzymatic process is now well accepted, some mechanistic issues are still debated. Using homogeneous E. coli enzyme we have shown that, contrary to previous report based on in vivo experiments, there is no exchange of the cylopropane methylene protons with the solvent during catalysis, as probed by ultra high resolution mass spectrometry. Using [methyl-14C]-labeled and [methyl- 3H3]-S-adenosyl-l-methionine we have measured a significant intermolecular primary tritium kinetic isotope effect (T V/Kapp =1.8±0.1) consistent with a partially rate determining deprotonation step. We conclude that both chemical steps of this enzymatic cyclopropanation, the methyl addition onto the double bond and the deprotonation step, are rate determining, a common situation in efficient enzymes. [ABSTRACT FROM AUTHOR]

Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7- epi -cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) ( K m  = 2.1 μM), and 2-oxoglutarate ( K m  = 3.2 μM), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1 μM. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) ( K D  = 0.02 μM) and 2-oxoglutarate ( K D  = 53 μM and K D  = 1.1 μM in the absence or presence of 10 μM Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87–108) seems to modulate the stereoselectivity of the reaction. [ABSTRACT FROM AUTHOR]

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to l-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 A resolution. AnaB is a homotetramer and its fold is very similar to that of the acyl- CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl- CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl- CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes. [ABSTRACT FROM AUTHOR]

Abstract: We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids. [Copyright &y& Elsevier]

Abstract: The neurotoxic l-2-amino-3-methylaminopropionic acid (BMAA) was hypothesized to be involved in sporadic cases of amyotrophic lateral sclerosis (ALS). Studies highlighting a possible implication of environmental factors in the incidence of sporadic ALS have become more numerous over recent years. Over the past years, the most widely used method for quantifying BMAA was based on the derivatization of this polar and basic molecule with a fluorescent compound (6-aminoquinolonyl-N-hydroxysuccinimidyl, 6-AQC). This derivatization allows the retention of the conjugate by reversed-phase liquid chromatography and its detection by fluorescence. Nevertheless, recent findings have shown that this method applied to complex samples may cause false positive responses. We therefore developed an analytical procedure for the determination of underivatized BMAA at trace level in complex environmental matrices (river water, cyanobacteria and biofilm) using solid-phase extraction (SPE) based on mixed mode sorbent to concentrate and clean up real samples. Analyzes were performed by hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization and tandem mass spectrometry used in multiple reaction monitoring scan mode. Analytical procedures were validated for the different natural samples using the total error approach. BMAA can be quantified by these reliable and highly selective analytical methods in a range of only a few ngmL−1 in river water and a few ngmg−1 dry weight in cyanobacteria and biofilm matrices. [Copyright &y& Elsevier]

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: We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC–MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain. [Copyright &y& Elsevier]

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins as well as the in-vitro reconstitution of the first steps of this biosynthesis. We now report experimental evidence supporting the proposed reaction mechanism of AnaB, a flavoprotein homologous to acyl-CoA dehydrogenase. AnaB catalyzes the two-electron oxidation of prolyl-AnaD, which is proline linked to the acyl carrier protein holo-AnaD, to dehydroprolyl-AnaD using oxygen as the second substrate. AnaB is thus an oxidase. By using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we have identified and characterized dehydroprolyl-AnaD, the AnaB product. We estimated an apparent catalytic constant of 1 min-1 for AnaB catalysis. We synthesized several deuterium-labeled prolines and enzymatically transformed them into their corresponding prolyl-AnaD. These deuterium-labeled prolyl-AnaDs were oxidized in the presence of AnaB, and the deuterium labeling in the remaining substrate and in the product was determined by LC-MS/MS. The data supported a reaction mechanism starting with a rapid enolization followed by a slow oxidation to give the conjugated imine, which in turn was isomerized to pyrroline-5-carboxyl-AnaD. We also showed that cis- and trans-4-fluoro-l-prolyl-AnaD and 3,4-dehydro-l-prolyl-AnaD were transformed into pyrrole-2-carboxyl-AnaD by AnaB. Thus, the 4-fluoro-analogues experienced a β-elimination supporting the AnaB-catalyzed aza-allylic isomerization. We identified by sequence alignment the AnaB active site base, Glu244. We produced, purified, and characterized the E244A AnaB mutant, which is inactive, supporting the catalytic role of E244 as a base. [ABSTRACT FROM AUTHOR]

Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillaforia sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 pg/mg and 0.29 j.μg/mg, respectively) and cylindrospermopsin (0.10 μg/mg and 0.11 rig/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 ± 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA. [ABSTRACT FROM AUTHOR]

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins, We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the Starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillaoria PCC 6506. We identified in PCC 6506 the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT, that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-prohne on holo-AnaD and was specific for L-proline (Km 0.97 mM, kcat = 68 min-1) among the 20 natural amino acids. Among six close structural analogues of L-proline, including (S)-l-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (Km = 1.5 mM, kcat 29 min-1). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD-containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an α-β-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE, [ABSTRACT FROM AUTHOR]

S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacteriurn 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 IC50 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. [ABSTRACT FROM AUTHOR]

We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins. [ABSTRACT FROM AUTHOR]

Abstract: 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 μM) and to the pyridoxamine 5′-phosphate form (K i2 = 1.7 ± 0.2 μM) 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 μM, and K i2 = 0.9 ± 0.2 μM. These findings pave the way to the design of new antimycobacterial drugs. [Copyright &y& Elsevier]

Abstract: Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC50 ranging from 1 to 10 µM, in the presence of 750 µM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K I =6 µM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 µM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme. [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]

Escherichia colifatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6-tagged protein. This recombinant enzyme is as active as the native enzyme with aKm of 90 µmforS-AdoMet and a specific activity of 5 × 10−2 µmol·min−1·mg−1. The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of the methyl protons ofS-AdoMet to the solvent, data that do not support the ylide mechanism. Inactivation of the enzyme by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of 1.2 m−1·s−1, is not protected by substrates. Graphical analysis of the inactivation by DTNB revealed that only one cysteine is responsible for the inactivation of the enzyme. The three strictly conserved Cys residues among cyclopropane synthases, C139, C176 and C354 of theE. colienzyme, were mutated to serine. The relative catalytic efficiency of the mutants were 16% for C139S, 150% for C176S and 63% for C354S. The three mutants were inactivated by DTNB at a rate comparable to the rate of inactivation of the His6-tagged wild-type enzyme, indicating that the Cys responsible for the loss of activity is not one of the conserved residues. Therefore, none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis. The inactivation is probably the result of steric hindrance, a phenomenon irrelevant to catalysis. It is very likely thatE. coliCFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified. [ABSTRACT FROM AUTHOR]

Analogues of S-adenosyl-l-methionine were synthesized and evaluated as inhibitors of the purified E. coli cyclopropane fatty acid synthase, a model for M. tuberculosis cyclopropane synthases that are potential targets for antituberculous drugs. Our results show that the presence of the adenosine moiety, in the inhibitor, is required for strong binding, but that the sulfonium charge is less important. The best inhibitors found were S-adenosyl-l-homocysteine and its sulfoxides. [Copyright &y& Elsevier]

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites. [ABSTRACT FROM AUTHOR]

The article provides a strong evidence on the successful identification of gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506. According to the authors, anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria. They reveal that they have identified a 29 kb DNA fragment containing a sequence called ks2 that previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a.