scholarly journals β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)

2001 ◽  
Vol 183 (11) ◽  
pp. 3526-3530 ◽  
Author(s):  
W. Peter Revill ◽  
Maureen J. Bibb ◽  
Ann-Karolin Scheu ◽  
Helen J. Kieser ◽  
David A. Hopwood
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Streptomyces Coelicolor ◽  
Mrna Transcript ◽  
Acid Biosynthesis ◽  
Biosynthesis Gene Cluster ◽  
Leaderless Mrna ◽  
Ketoacyl Synthase ◽  
Biosynthesis Gene

ABSTRACT The Streptomyces coelicolor fab (fatty acid biosynthesis) gene cluster (fabD-fabH-acpP-fabF) is cotranscribed to produce a leaderless mRNA transcript. One of these genes, fabH, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in S. coelicolor.

scholarly journals Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

2005 ◽  
Vol 187 (11) ◽  
pp. 3795-3799 ◽  
Author(s):  
Yongli Li ◽  
Galina Florova ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Streptomyces Coelicolor ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
E Coli ◽  
Cell Extracts ◽  
Branched Chain

ABSTRACT The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (∼70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a ΔfabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

Planta ◽  
2010 ◽  
Vol 231 (6) ◽  
pp. 1277-1289 ◽  
Author(s):  
Damián González-Mellado ◽  
Penny von Wettstein-Knowles ◽  
Rafael Garcés ◽  
Enrique Martínez-Force
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Acid Biosynthesis

scholarly journals Recognition of Intermediate Functionality by Acyl Carrier Protein over a Complete Cycle of Fatty Acid Biosynthesis

2010 ◽  
Vol 17 (7) ◽  
pp. 776-785 ◽  
Author(s):  
Eliza Płoskoń ◽  
Christopher J. Arthur ◽  
Amelia L.P. Kanari ◽  
Pakorn Wattana-amorn ◽  
Christopher Williams ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Complete Cycle

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.

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