In-vitro evidence for feed-back regulation of ?-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis

Planta ◽  
1996 ◽  
Vol 198 (2) ◽  
Author(s):  
FritziM. Br�ck ◽  
Monika Brummel ◽  
Ricardo Schuch ◽  
Friedrich Spener
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Medium Chain

scholarly journals Engineered Fatty Acid Biosynthesis inStreptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

2001 ◽  
Vol 183 (7) ◽  
pp. 2335-2342 ◽  
Author(s):  
Natalya Smirnova ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
Branched Chain

ABSTRACT The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D3 acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.

scholarly journals β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

2000 ◽  
Vol 182 (2) ◽  
pp. 365-370 ◽  
Author(s):  
Keum-Hwa Choi ◽  
Richard J. Heath ◽  
Charles O. Rock
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Biochemical Basis ◽  
E Coli ◽  
Branched Chain

ABSTRACT A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the β-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in theBacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. colienzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

scholarly journals Condensing enzymes fromCuphea wrightiiassociated with medium chain fatty acid biosynthesis

1998 ◽  
Vol 13 (5) ◽  
pp. 611-620 ◽  
Author(s):  
Mary B. Slabaugh ◽  
Jeffrey M. Leonard ◽  
Steven J. Knapp
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Acid Biosynthesis ◽  
Medium Chain

β-Ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds

Planta ◽  
2002 ◽  
Vol 215 (5) ◽  
pp. 847-854 ◽  
Author(s):  
Burkhardt Schütt ◽  
Amine Abbadi ◽  
Brigitte Loddenkötter ◽  
Monika Brummel ◽  
Friedrich Spener
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Carrier Protein ◽  
Medium Chain ◽  
Key Enzyme ◽  
Cuphea Lanceolata

scholarly journals Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

1998 ◽  
Vol 180 (17) ◽  
pp. 4481-4486 ◽  
Author(s):  
Lei Han ◽  
Sandra Lobo ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chain Fatty Acid ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Streptomyces Glaucescens

ABSTRACT The Streptomyces glaucescens fabH gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed anM r of 37,000, while gel filtration analysis determined a native M r of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a Km value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the S. glaucescensFabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.

scholarly journals Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

2015 ◽  
Vol 6 ◽  
Author(s):  
Kyle B. Reynolds ◽  
Matthew C. Taylor ◽  
Xue-Rong Zhou ◽  
Thomas Vanhercke ◽  
Craig C. Wood ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Metabolic Engineering ◽  
Nicotiana Benthamiana ◽  
Fatty Acid Biosynthesis ◽  
Chain Fatty Acid ◽  
Benthamiana Plant ◽  
Medium Chain Fatty Acid ◽  
Acid Biosynthesis ◽  
Plant Leaf ◽  
Medium Chain
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