Structural stability of Cutibacterium acnes acyl carrier protein studied using CD and NMR spectroscopy

AbstractTo survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.

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Molecular basis for the substrate specificity of quorum signal synthases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705400114 ◽

2017 ◽

Vol 114 (34) ◽

pp. 9092-9097 ◽

Cited By ~ 27

Author(s):

Shi-Hui Dong ◽

Nicole D. Frane ◽

Quin H. Christensen ◽

E. Peter Greenberg ◽

Rajesh Nagarajan ◽

...

Keyword(s):

Fatty Acids ◽

Substrate Specificity ◽

Molecular Basis ◽

Acyl Carrier Protein ◽

Acyl Donor ◽

Carrier Protein ◽

Homoserine Lactones ◽

Acyl Homoserine Lactones ◽

Signal Synthesis ◽

Enzyme Complexes

In severalProteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl–homoserine lactones (AHL) signals usingS-adenosyl–l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA–aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure–function, and kinetic analysis define the features that facilitate the unusual acyl conjugation withS-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.

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Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH)

Biotechnology and Bioengineering ◽

10.1002/bit.25583 ◽

2015 ◽

Vol 112 (8) ◽

pp. 1613-1622 ◽

Cited By ~ 11

Author(s):

Wen Jiang ◽

Yanfang Jiang ◽

Gayle J. Bentley ◽

Di Liu ◽

Yi Xiao ◽

...

Keyword(s):

Fatty Acids ◽

Acyl Carrier Protein ◽

Carrier Protein ◽

Enhanced Production ◽

Branched Chain Fatty Acids ◽

Branched Chain ◽

Chain Fatty Acids

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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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A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat's Claw Seed

PLANT PHYSIOLOGY ◽

10.1104/pp.117.2.593 ◽

1998 ◽

Vol 117 (2) ◽

pp. 593-598 ◽

Cited By ~ 66

Author(s):

Edgar B. Cahoon ◽

Salehuzzaman Shah ◽

John Shanklin ◽

John Browse

Keyword(s):

Substrate Specificity ◽

Acyl Carrier Protein ◽

Carrier Protein

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Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids

The Plant Journal ◽

10.1111/j.1365-313x.2005.02553.x ◽

2005 ◽

Vol 44 (4) ◽

pp. 620-632 ◽

Cited By ~ 39

Author(s):

Abraham J. K. Koo ◽

Martin Fulda ◽

John Browse ◽

John B. Ohlrogge

Keyword(s):

Fatty Acids ◽

Acyl Carrier Protein ◽

Carrier Protein

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

Journal of Bacteriology ◽

10.1128/jb.182.2.365-370.2000 ◽

2000 ◽

Vol 182 (2) ◽

pp. 365-370 ◽

Cited By ~ 173

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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A universal pocket in Fatty acyl-AMP ligases ensures redirection of fatty acid pool away from Coenzyme A-based activation

eLife ◽

10.7554/elife.70067 ◽

2021 ◽

Vol 10 ◽

Author(s):

Gajanan Shrikant Patil ◽

Priyadarshan Kinatukara ◽

Sudipta Mondal ◽

Sakshi Shambhavi ◽

Ketan D Patel ◽

...

Keyword(s):

Fatty Acids ◽

Binding Sites ◽

Molecular Basis ◽

Coenzyme A ◽

Acyl Carrier Protein ◽

Fatty Acyl ◽

Carrier Protein ◽

Binding Pockets ◽

Fatty Acid Pool ◽

Do So

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3', 5'-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organisations. The universal distribution of FAALs suggests they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

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