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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]

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]

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]

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.

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.

Introduction: Amyotrophic lateral sclerosis (ALS) is the most common motor neurone disease. It occurs in two forms: (1) familial cases, for which several genes have been identified and (2) sporadic cases, for which various hypotheses have been formulated. Notably, the β-N-methylamino-L-alanine (L-BMAA) toxin has been postulated to be involved in the occurrence of sporadic ALS. The objective of the French BMAALS programme is to study the putative link between L-BMAA and ALS., Methods and Analysis: The programme covers the period from 1 January 2003 to 31 December 2011. Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this period in the area under study (10 counties spread over three French regions) were collected. First, the standardised incidence ratio will be calculated for each municipality under concern. Then, by applying spatial clustering techniques, overincidence and underincidence zones of ALS will be sought. A case-control study, in the subpopulation living in the identified areas, will gather information about patients' occupations, leisure activities and lifestyle habits in order to assess potential risk factors to which they are or have been exposed. Specimens of drinking water, food and biological material (brain tissue) will be examined to assess the presence of L-BMAA in the environment and tissues of ALS cases and controls., Ethics and Dissemination: The study has been reviewed and approved by the French ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-Mer IV). The results will be published in peer-reviewed journals and presented at national and international conferences., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

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/K(app)=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., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)