Escherichia coli cyclopropane fatty acid synthase.

Escherichia coli fatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6-tagged protein. This recombinant enzyme is as active as the native enzyme with a Km of 90 microm for S-AdoMet and a specific activity of 5 x 10(-2) micromol.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 of S-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 the E. coli enzyme, 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 that E. coli CFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified.     

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.     

Identification of new inhibitors of E. coli cyclopropane fatty acid synthase using a colorimetric assay

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: Km (AdoMet)=80 microM, kcat=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 microM and 11 microM, respectively. This new assay will thus be useful for high-throughput screening of compound libraries for discovering novel antituberculous drug candidates.     

Cryosensitivity of Escherichia coli and the involvement of cyclopropane fatty acids

C alcott , P.H. O liver , J.D. D ickey , K. & C alcott K atherine , 1984. Cryosensitivity of Escherichia coli and the involvement of cyclopropane fatty acids. Journal of Applied Bacteriology 56 , 165–172.
Strains of Escherichia coli proficient and deficient in cylopropane fatty acid synthesis were compared for fatty acid content, cryosensitivity, presence of freeze-thaw-induced wall and membrane damage, resistance to detergent-stimulated lysis and tolerance to salt and detergents during growth. The mutant populations synthesized much less cyclopropane fatty acids and were more resistant than the wild type to freezing and thawing in saline only, exhibiting less viability loss and less wall and membrane damage. While the resistance of the mutants to NaCl was unaltered, their detergent resistance was decreased under both growth and non-growth conditions. Although these physiological changes were associated with a lower cyclopropane fatty acid content in the mutant strains, it is proposed that the responses were due to the altered membrane fluidity of the mutants due to changes in their unsaturated fatty acid content.     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

Some factors affecting cyclopropane acid formation in Escherichia coli

1. The fatty acid composition of the extractable lipids of Escherichia coli varied with growth conditions. 2. The principal fatty acids were palmitic acid, hexadecenoic acid, octadecenoic acid and the cyclopropane acids, methylenehexadecanoic acid and methyleneoctadecanoic acid. 3. Cyclopropane acid formation from monoenoic acids was increased by acid media, poor oxygen supply, or high growth temperature. 4. Cyclopropane acid formation was decreased by alkaline media, well oxygenated conditions, the presence of citrate, or lack of Mg(2+).     

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci

Summary The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.     

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82 g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53 g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.     

Inhibition of cyclopropane fatty acid synthase by sinefungin and A9145C

Sinefungin and A9145C, antifungal antibiotic analogs of S-adeno-sylmethionine isolated from $\text{Streptomyces}\text{,}\text{griseolus}$, have been found to be very effective $\text{in}\text{,}\text{vitro}$ inhibitors of cyclopropane fatty acid synthase from $\text{Lactobacillus}\text{,}\text{plantarum}$. Both compounds exhibit linear competitive inhibition with a K_i for Sinefungin of 220 nM and a K_i for A9145C of 11 nM.     

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.     

Synthesis and evaluation of analogues of S-adenosyl-L-methionine, as inhibitors of the E. coli cyclopropane fatty acid synthase

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.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Amino acid sequence of Escherichia coli citrate synthase

Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented. The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously [Ner, S. S., Bhayana, V., Bell, A. W., Giles, I. G., Duckworth, H. W., & Bloxham, D. P. (1983) Biochemistry 22, 5243]; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10. Substantial regions of sequence homology are noted between the E. coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site. Deletions or insertions must be assumed in a number of places in order to maximize homology. Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated. It appears that E. coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E. coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.