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

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

Journal of Bacteriology ◽

10.1128/jb.187.11.3795-3799.2005 ◽

2005 ◽

Vol 187 (11) ◽

pp. 3795-3799 ◽

Cited By ~ 45

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.

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

Journal of Bacteriology ◽

10.1128/jb.183.7.2335-2342.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2335-2342 ◽

Cited By ~ 23

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.

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Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

10.21203/rs.3.rs-72626/v1 ◽

2020 ◽

Author(s):

Michael Burkart ◽

Thomas Bartholow ◽

Terra Sztain ◽

Ashay Patel ◽

D Lee ◽

...

Keyword(s):

Fatty Acid ◽

Protein Interactions ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Acyl Carrier Protein ◽

Protein Docking ◽

Carrier Protein ◽

Protein Protein Interactions ◽

Acid Biosynthesis ◽

E Coli

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.

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Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis

Biochemical Journal ◽

10.1042/bj3210313 ◽

1997 ◽

Vol 321 (2) ◽

pp. 313-318 ◽

Cited By ~ 4

Author(s):

Elke WINTER ◽

Monika BRUMMEL ◽

Ricardo SCHUCH ◽

Friedrich SPENER

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Condensation Reaction ◽

Simultaneous Increase ◽

Carrier Protein ◽

Acid Biosynthesis ◽

Cuphea Lanceolata ◽

Reducing Equivalents

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalents NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.

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Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase

Planta ◽

10.1007/bf00197585 ◽

1996 ◽

Vol 198 (1) ◽

Cited By ~ 37

Author(s):

VictoriaS. Eccleston ◽

AnnM. Cranmer ◽

ToniA. Voelker ◽

JohnB. Ohlrogge

Keyword(s):

Fatty Acid ◽

Brassica Napus ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Chain Fatty Acid ◽

Medium Chain Fatty Acid ◽

Carrier Protein ◽

Acid Biosynthesis ◽

Medium Chain

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

Journal of Bacteriology ◽

10.1128/jb.180.17.4481-4486.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4481-4486 ◽

Cited By ~ 68

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.

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