scholarly journals Plant holo-(acyl carrier protein) synthase

1988 ◽  
Vol 252 (1) ◽  
pp. 39-45 ◽  
Author(s):  
S A Elhussein ◽  
J A Miernyk ◽  
J B Ohlrogge
Keyword(s):  
Spinacia Oleracea ◽  
Acyl Carrier Protein ◽  
Ricinus Communis ◽  
Plant Cells ◽  
Gel Permeation Chromatography ◽  
Carrier Protein ◽  
Improved Method ◽  
Ph Optimum ◽  
Spinach Leaf

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3′,5′-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

scholarly journals Site-specific serine phosphorylation of spinach leaf sucrose-phosphate synthase

1992 ◽  
Vol 283 (3) ◽  
pp. 877-882 ◽  
Author(s):  
J L A Huber ◽  
S C Huber
Keyword(s):  
Okadaic Acid ◽  
Spinacia Oleracea ◽  
Sucrose Phosphate Synthase ◽  
Serine Phosphorylation ◽  
Spinacia Oleracea L ◽  
Spinach Leaf ◽  
Sucrose Phosphate ◽  
Phosphate Synthase

We recently reported [Huber, Huber & Nielsen (1989) Arch. Biochem. Biophys. 270, 681-690] that spinach (Spinacia oleracea L.) sucrose-phosphate synthase (SPS; EC 2.4.1.14) was phosphorylated in vivo when leaves were fed [32P]Pi. In vitro the enzyme was phosphorylated and inactivated by using [gamma-32P]ATP. We now report that SPS is phosphorylated both in vivo and in vitro on serine residues. The protein is phosphorylated at multiple sites both in vivo and in vitro as indicated by two-dimensional peptide maps of the immunopurified SPS protein. After being fed with radiolabel, leaves were illuminated or given mannose (which activates the enzyme), in the presence or absence of okadaic acid. Feeding okadaic acid to leaves decreased the SPS activation state in the dark and light and in leaves fed mannose. Across all the treatments, the activation state of SPS in situ was inversely related to the labelling of two phosphopeptides (designated phosphopeptides 5 and 7). These two phosphopeptides are phosphorylated when SPS is inactivated in vitro with [gamma-32P]ATP, and thus are designated as regulatory (inhibitory) sites [Huber & Huber (1991) Biochim. Biophys. Acta 1091, 393-400]. Okadaic acid increased the total 32P-labelling of SPS and in particular increased labelling of the two regulatory sites, which explains the decline in activation state. In the presence of okadaic acid, two cryptic phosphorylation sites became labelled in vivo that were not apparent in the absence of the inhibitor. Overall, the results suggest that light/dark regulation of SPS activity occurs as a result of regulatory serine phosphorylation. Multiple sites are phosphorylated in vivo, but two sites in particular appear to regulate activity and dephosphorylation of these sites in vivo is sensitive to okadaic acid.

scholarly journals In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid

2004 ◽  
Vol 48 (1) ◽  
pp. 242-249 ◽  
Author(s):  
Stéphanie Ducasse-Cabanot ◽  
Martin Cohen-Gonsaud ◽  
Hedia Marrakchi ◽  
Michel Nguyen ◽  
Didier Zerbib ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Dynamic Equilibrium ◽  
Acyl Carrier Protein ◽  
Multidrug Resistant ◽  
Carrier Protein ◽  
Ligand Complex ◽  
Fas Ii

ABSTRACT The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Escherichia coliLipA Is a Lipoyl Synthase:  In Vitro Biosynthesis of Lipoylated Pyruvate Dehydrogenase Complex from Octanoyl-Acyl Carrier Protein†

Biochemistry ◽  
2000 ◽  
Vol 39 (49) ◽  
pp. 15166-15178 ◽  
Author(s):  
J. Richard Miller ◽  
Robert W. Busby ◽  
Sean W. Jordan ◽  
Jennifer Cheek ◽  
Timothy F. Henshaw ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Acyl Carrier Protein ◽  
Pyruvate Dehydrogenase Complex ◽  
Carrier Protein ◽  
In Vitro Biosynthesis ◽  
Lipoyl Synthase ◽  
Dehydrogenase Complex

scholarly journals Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

1996 ◽  
Vol 40 (12) ◽  
pp. 2813-2819 ◽  
Author(s):  
R A Slayden ◽  
R E Lee ◽  
J W Armour ◽  
A M Cooper ◽  
I M Orme ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Type I ◽  
Dependent Manner ◽  
Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

scholarly journals RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids in Pseudomonas aeruginosa

2008 ◽  
Vol 190 (9) ◽  
pp. 3147-3154 ◽  
Author(s):  
Kun Zhu ◽  
Charles O. Rock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Molecular Ruler ◽  
Necessary And Sufficient

ABSTRACT Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of β-hydroxydecanoyl-β-hydroxydecanoate from two molecules of β-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily β-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for β-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

scholarly journals Characterization of a Novel Acyl Carrier Protein, RkpF, Encoded by an Operon Involved in Capsular Polysaccharide Biosynthesis in Sinorhizobium meliloti

1998 ◽  
Vol 180 (18) ◽  
pp. 4950-4954 ◽  
Author(s):  
Guido Epple ◽  
Koen M. G. M. van der Drift ◽  
Jane E. Thomas-Oates ◽  
Otto Geiger
Keyword(s):  
Sinorhizobium Meliloti ◽  
Capsular Polysaccharide ◽  
Acyl Carrier Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Product ◽  
Spectrometric Analysis ◽  
Carrier Protein ◽  
Plant Host

ABSTRACT Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in Sinorhizobium meliloti AK631 functionalrkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpressed and purified the derived protein product (RkpF) in Escherichia coli. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive β-alanine added to the growth medium. If homogeneous RkpF protein is incubated with radiolabeled coenzyme A in the presence of purified holo-ACP synthase from E. coli, an in vitro transfer of 4′-phosphopantetheine to the RkpF protein can be observed. The conversion from apo-RkpF protein to holo-RkpF protein seems to go along with a major conformational change of the protein structure, because the holo-RkpF protein runs significantly faster on native polyacrylamide gel electrophoresis than the apo-RkpF protein. Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF protein and a mass of 9,927 for the holo-RkpF protein. Our data show that RkpF is a novel ACP.

Sign in / Sign up

Export Citation Format

Share Document