Purification of FabF1 and FabZ Plasmid pHW76 and pHW74m were introduced into strain BL21 (DE3), respectively, and the proteins were overexpressed and purified as described previously[20]. The enzymes were homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The E. coli FabD, FabH, FabG, FabA, FabZ, FabB,

FabI and Vibrio harveyi AasS proteins were purified by their hexahisitidine tags described previously [18, 20]. Assay of FabF1 and FabZ activity in vitro Fatty acid synthesis https://www.selleckchem.com/products/AZD1152-HQPA.html was reconstituted in vitro to assay FabF1 and FabZ activity using the purified enzymes that catalyze the fatty acid biosynthesis essentially. The assay utilized the AasS acyl-ACP synthetase from Vibrio harveyi [18] to generate 3-hydroxydecanoyl-ACP. The reaction mixtures to synthesize 3-hydroxydecanoyl-ACP contained 20 μM ACP, 10 mM click here ATP, 10 mM MgSO4, 5 mM DTT, 0.1 M sodium phosphate buffer (pH 7.0), 100 μM 3-hydroxydecanoic acid

(Sigma) and AasS (0.2 μg) in a final volume of 50 μl. The mixtures were incubated at 37°C for 1 h. To assay C. acetobutylicium FabF1, the following incubation 1 μg each of E. coli FabD, FabG and FabA, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of either E. coli FabB or C. acetobutylicium FabF1 was added. To assay C. acetobutylicium FabZ, the following incubation contained 1 μg each of E. coli FabD, FabG and FabB, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of E. coli FabA or C. acetobutylicium FabZ was added. The resulting mixture was incubated for an additional 1 h and the reaction products were analyzed by conformationally sensitive gel electrophoresis on 20% polyacrylamide gels containing 2.5 M urea [20, 24]. The gel was stained with Coomassie Brilliant Blue R250. Acknowledgements This work was supported by the President Foundation of South China Agricultural University and NIH next grant AI15650. We are grateful to Professor Hiroshi Kobayashi (Selleckchem Selonsertib Graduate School of Pharmaceutical Sciences, Chiba University Japan) for critical reading. Electronic supplementary material Additional file 1: Bacterial strains, plasmids and oligonucleotides used in this work. The data provided bacteria strains,

plasmids and oligonucleotides used in this work. (PDF 104 KB) References 1. Cronan JE: Bacterial membrane lipids: where do we stand? Annu Rev Microbiol 2003, 57:203–224.CrossRefPubMed 2. Mansilla MC, de Mendoza D: The Bacillus subtilis desaturase: a model to understand phospholipid modification and temperature sensing. Archives of microbiology 2005,183(4):229–235.CrossRefPubMed 3. Bloch K, Baronowsky P, Goldfine H, Lennarz WJ, Light R, Norris AT, Scheuerbrandt G: Biosynthesis and metabolism of unsaturated fatty acids. Fed Proc 1961, 20:921–927.PubMed 4. Scheuerbrandt G, Goldfine H, Baronowsky PE, Bloch K: A novel mechanism for the biosynthesis of unsaturated fatty acids. J Biol Chem 1961, 236:PC70-PC71.PubMed 5. Bloch K: Beta-Hydroxythioester dehydrase.