Acyl carrier protein

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 the Bacillus 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 ...

The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoylcompounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.

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...

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-"%C]acetyl-CoA-labelled Cys""", and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys"""Ser mutant, however, still Abbreviations used : KAS, 3-oxoacyl-(acyl-carrier-protein) synthase ; ACP, acyl carrier protein ; FAS, fatty acid synthase. 1 To whom correspondence should be addressed (e-mail spener!uni-muenster.de). was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His#'" with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His#'". Double mutants Cys"""Ser\His#'"Ala and Cys"""Ser\His#'"Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-"%C]malonyl-ACP, a reaction indicating the role of His#'" in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg"&! and Arg$!' in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

Mitochondria are intracellular organelles located in the cytoplasm, that are essential for life and health. It is also known as the powerhouse of the cells and use a process called oxidative phosphorylation to generate energy. In side mitochondria, the lipid monomer fatty acids act as energy sources, membrane constituents, molecules for post translational modifications of proteins and cellular signaling factors. Plant cells possess couple of distinct type 2 Fatty Acid Synthase (FAS) systems. The two systems take place in plastids (ptFAS) and mitochondria (mtFAS). Lots of mtFAS components have been identified but, the physiological functions of mtFAS are still poorly understood. In this review, we have characterized five Arabidopsis mtFAS components, namely Mitochondrial β-Ketoacyl-ACP Synthase (mtKAS), Mitochondrial 3-Hydroxyacyl-ACP Dehydratase (mtHD), Mitochondrial Enoyl-ACP reductase (mtER), Mitochondrial Phosphopantetheinyl Transferase (mtPPT), and Mitochondrial Malonyl-CoA Synthetase (mtMCS). MtKAS, mtHD, and mtER catalyze 3 reactions of the 4 reactions of the acyl chain elongation cycle; mtPPT is responsible for activating Mitochondrial Acyl Carrier Protein (mtACP) by the addition of a phosphopantetheine cofactor; and mtMCS generates malonyl-CoA, the donor of 2-carbon elongation unit for the mtFAS system. Mitochondrial octanoyl-ACP is the precursor for the synthesis of lipoic acid, which is the coenzyme of the H-subunit of Glycine Decarboxylase Complex (GDC), a key enzyme of photorespiration. Accordingly, mutations in the mtFAS system can lead to a typical deficiency in photorespiration due to the depletion of the lipoylation of the H-subunit of GDC. These mutants are altered as an inferior effect of the deficiency in photorespiration. In addition, mutant analyses demonstrate that mitochondrial 3-hydroxymyristyl-ACP contributes to the biosynthesis of lipid alike molecules, suggesting a novel synthetic destination of the mtFAS intermediates. RNA-seq transcriptomic analyses of the mutants establish a regulatory network that is associated with the mtFAS functions is crucial for plant metabolic homeostasis.

During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1- mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions o...

Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differin...

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (BnlO) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seedexpressed lipid synthesis genes are regulated by separate tissuespecific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter adivity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P.

The nucleotide sequence is reported for a cDNA containing the entire coding region of a stearoyl-ACP desaturase (EC 1.14.99.6) from Brassica napus L. cv. Jet neuf. The cDNA was obtained from a library constructed from poly(A) + RNA purified from embryo tissue. The derived amino acid sequence demonstrates substantial similarity with those from other plant A9-desaturases. Comparative RNA-dot blot analyses using the A9-desaturase cDNA and a rapeseed oleosin cDNA as probes showed that although both these transcripts were seed-specific, they exhibited distinct patterns of temporal regulation. The desaturase message was induced by 25 days after anthesis (DAA), peaking at 45 DAA but decreasing considerably thereafter. In contrast, the oleosin transcript did not increase until 45-50 DAA, reaching a peak much later at about 70 DAA.

Highlights.  β-ketoacyl-acyl carrier protein synthase I (FabB) may be one of the most attractive biochemical pathways to be used as the target for new antibacterial agents.  Flavonoids are naturally occurring polyphenolic compounds that they have been reported to possess many useful properties, including anti-inflammatory activity, oestrogenic activity, enzyme inhibition, antimicrobial activity.  We suggest two flavonoid compounds ,Genistein and Isorhamnetin, can be used as a hit molecule against the fatty acid synthase in bacteria.  Genistein and Isorhamnetin, have similar binding poses and interactions with β-ketoacyl acyl carrier protein synthase I compared to the standard (Thiolactomycin).  This in silico study is actually an added advantage to screen the β-ketoacyl acyl carrier protein synthase I inhibition.

Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches. Genome scale metabolic networks Á Applications of flux balance analysis Á Flux profiles Á Robustness through metabolic adjustment Á Incorporating gene expression profiles Abbreviations INH Isoniazid Mtb Mycobacterium tuberculosis InhA NADH-dependent enoyl [acyl-carrier-protein] reductase NAD(P) Nicotinamide adenine dinucleotide (phosphate) Ashwini G. Bhat and Rohit Vashisht contributed equally to this work.

In nonribosomal biosynthesis of peptide antibiotics by multimodular synthetases, amino acid monomers are activated by the adenylation domains of the synthetase and loaded onto the adjacent carrier protein domains as thioesters, then the formation of peptide bonds and translocation of the growing chain are effected by the synthetase's condensation domains. Whether the condensation domains have any editing function has been unknown. Synthesis of aminoacyl–coenzyme A (CoA) molecules and direct enzymatic transfer of aminoacyl-phosphopantetheine to the carrier domains allow the adenylation domain editing function to be bypassed. This method was used to demonstrate that the first condensation domain of tyrocidine synthetase shows low selectivity at the donor residue ( d -phenylalanine) and higher selectivity at the acceptor residue ( l -proline) in the formation of the chain-initiating d -Phe- l -Pro dipeptidyl-enzyme intermediate.

Acyl carrier protein (ACp) domains act as interaction hubs within modular polyketide synthase (pKs) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular pKs that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACp segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACp topology in both cases. this article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. the interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase pKs chain-extension modules. the polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACp-mediated substrate delivery within and between pKs modules.

Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward.

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also ...

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)-dependent condensation of D-pantoate and β-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD + by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent. (

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and ␤-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radiolabeled fatty acids in cultured procyclic parasites with [ 14 C]pyruvate or [ 14 C]threonine, either of which is catabolized to [ 14 C]acetyl-CoA in the mitochondrion. Although some of the [ 14 C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and β-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [14C]pyruvate or [14C]threonine, either of which is catabolized to ...

Quorum sensing is an intercellular mechanism used by many bacterial pathogens to regulate behaviors, such as motility, virulence, and antibiotic production. Disruption of quorum sensing is shown to be deleterious to bacterial pathogenicity, without causing lethality. In Gram-negative bacteria, acyl-homoserine lactone (AHL or acyl-HSL) signal molecules are synthesized by AHL synthase enzymes using acylated-acyl carrier protein (acyl-ACP) and S-adenosyl-L-methionine (SAM) as their substrates. Pantoea stewartii EsaI, which causes Stewart's wilt in corn, and Yersinia pestis YspI, which causes bubonic plague are AHL synthases that use 3-oxohexanoyl-ACP and 3-oxooctanoyl-ACP as the acyl-substrate to synthesize 3-oxohexanoyl-HSL and 3-oxooctanoyl-HSL signals, respectively. Unfortunately, the instability of the β-ketoacyl-ACP substrate due to the enolate formation at the -position in the acyl-chain impedes mechanistic investigations for β-ketoacyl-ACP utilizing enzymes. In this thesis, we designed, developed, and evaluated a library of stable β-ketoacyl-ACP mimics for two β-ketoacyl-ACP utilizing AHL synthases, EsaI and YspI. We found that 2-furanacetyl-ACP and 2-benzofuranacetyl-ACP were the best 3-oxoacyl-ACP alternative substrate for EsaI and YspI, respectively, and within an order of magnitude to that observed for well-characterized ACP/CoAdependent AHL synthases with their native acyl-ACP/ acyl-CoA substrate, such as RhlI, BmaI, and BjaI. The presence of a heteroatom other than oxygen is crucial to retain enzyme activity in both EsaI and YspI. Also, substrate inhibition of 2-thiopheneacetyl-ACP was observed with both enzymes. The success of synthesis and high activity of β-ketoacyl-ACP viii mimics for EsaI and YspI should open new doors in characterizing this class of enzymes. Moreover, the β-ketoacyl-ACP substrates could be used as chemical probes to explore and design inhibitors for therapeutically important AHL synthases and several uncharacterized enzymes that impacts human health, such as -ketoacyl-ACP reductase in fatty acid biosynthesis and polyketide synthase in polyketide synthesis which are targets for antimicrobial, antimalarial, and anti-cancer drugs. ix TABLE OF CONTENTS DEDICATION .

SummaryPolyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme‐bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti‐GFP antibody were observed, no novel compound was detected. 6‐methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS wi...

The X-ray structure determination at 2.4 A ˚resolution of the putative orsellinic acid C3 O-methyltransferase (CalO1) involved in calicheamicin biosynthesis is reported. Comparison of CalO1 with a homology model of the functionally related calicheamicin orsellinic acid C2 O-methyltransferase (CalO6) implicates several residues that are likely to contribute to the regiospecificity of alkylation. Consistent with the proposed requirement of an acyl-carrier-proteinbound substrate, this structural study also reveals structural determinants within CalO1 that are anticipated to accommodate an association with an acyl carrier protein.

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510–512] had higher rates of HCO3−-dependent oxygen evolution (up to 369μmol/h per mg of chlorophyll) and higher rates of [1-14C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H14CO3− in assays permitting high rats of photosynthesis. Incorporation of H14CO3− into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3–4 times in excess of that required to account for rates of [1-14C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloropl...

2 The abbreviations used are: ACP, acyl carrier protein; 4-PP, 4Ј-phosphopantetheine; MES 2-(N-morpholino)ethanesulfonic acid; DTT, dithiothreitol; cat, chloramphenicol acetyltransferase; Ni-NTA, nickel-nitrilotriacetic acid. 3 D. Keating and J. Cronan, unpublished data. 4 T. Stachelhaus and C. Walsh, personal communication.

Background: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-A2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADHdependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADHdependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus). Results: The crystal structure of an ENR-NAD binary complex has been determined at 1.9 A resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel 3 sheet flanked by seven at helices. The subunit has a topology highly reminiscent of a dinucleotidebinding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. Conclusions: The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3a,203-hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base -with subtle modifications arising from differing requirements of the reduction chemistry.

The NADP-dependent β-keto acyl-carrier protein reductase (BKR) from Brassica napus has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol of average molecular weight 1500 as the precipitant. The crystals belong to the hexagonal space group P6422, with unit-cell parameters a = b = 129.9, c = 93.1 Å, α = β = 90, γ = 120°. Calculated values for Vm , the use of rotation and translation functions and consideration of the packing suggest that the asymmetric unit contains a monomer. The crystals diffract to beyond 2.8 Å resolution and are more amenable to X-ray diffraction analysis than those reported previously for the Escherichia coli enzyme. The structure determination of B. napus BKR will provide important insights into the catalytic mechanism of the enzyme and into the evolution of the fatty-acid elongation cycle by comparisons with the other oxidoreductase of the pathway, enoyl acyl-carrier protein reductase (ENR).

The coding sequence of Δ9‐stearoyl‐(acyl carrier protein) desaturase from Ricinus communis was introduced into sunflower, under the control of seed‐specific promoter and terminator sequences of the late embryogenesis abundant gene from sunflower, Hads10. Two independent primary transformants contained three and six copies of the T‐DNA, as demonstrated by hybridization using nptII as a probe. The transgene proved genetically stable and was transmitted as a Mendelian trait. Transcript analysis of the heterologous Δ9‐stearoyl‐(acyl carrier protein) desaturase under control of the Hads10 promoter verified tissue‐specific expression in the developing embryos and not in the leaves. Fatty acid composition of the seed oil was followed over five generations under greenhouse and open field conditions. Some of the transgenic lines produced oil with a significantly reduced stearic acid content compared with non‐transformed plants under greenhouse and field conditions. However, additional studie...

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to la...

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 the Bacillus 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 ...

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA: AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.

. Ž . Transcripts from immature fibers and stripped ovules fibers removed of cotton Gossypium hirsutum L. were compared by differential display to identify cDNA fragments that represent mRNAs that are expressed primarily in cotton fibers. Eight independent fiber-specific cDNA fragments were isolated. One of these cDNAs had strong sequence similarity Ž . with acyl carrier protein ACP . A full-length cDNA for the cotton fiber-specific ACP was isolated using a PCR cDNA library screening technique. This 713 bp cDNA has an open reading frame that encodes a 136 amino acid polypeptide. Overall nucleotide and amino acid sequence identities with other plant ACP gene sequences averaged 66% and 60% respectively. A 19 amino acid sequence surrounding the prosthetic group attachment site is nearly identical to other plant ACP genes. Northern blot analyses showed that transcripts homologous to this fiber-specific ACP cDNA were predominantly expressed during the elongation stage of fiber development. Initial genomic Southern blot analysis indicated that a single copy of the fiber-specific ACP gene may be present in both the cotton A and D genomes, since diploid Gossypium species with A or D genomes gave identical bands. We speculate that this putative fiber-specific ACP may play an important role in rapidly elongating cotton fibers by contributing to the synthesis of membrane lipids. It is also apparent that during the evolution of cotton a member of the ACP gene family has been recruited for specific expression in cotton fibers.

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N -acyl- l -homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the Gram-negative bacterium Burkholderia glumae , QS controls virulence, motility, and protein secretion and is mediated by the binding of N -octanoyl- l -HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8...

Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles (“thiopyridines”), inhibited both purified ENR (50% inhibitory concentration [IC 50 ] = 3 to 25 μM) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 μg/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [ 14 C]acetate into fatty acids ...

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to ...

LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent Nacylation of UDP-3-O-(acyl)-α-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 Å crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel β-helix (LβH), and a C-terminal α-helical domain. The backbones of the LβH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LβH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LβH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120°r otation of the C-terminal domain relative to the LβH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 °C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2′. LpxD catalyzes the third step of lipid A biosynthesis, the acyl-ACP dependent N-acylation of the intermediate UDP-3-O-(acyl)-α-D-glucosamine (Scheme 1) (1-4). LpxD is conserved among all Gram-negative bacteria that make lipid A and is required for growth (1-4). It is therefore an excellent target for the development of new antibiotics. Three related crystal structures of LpxD from Chlamydia trachomatis (CtLpxD) 1 have recently been reported by Buetow et al. (5): an "apo" structure at 2.7 Å resolution, and two structures with bound UDP-N-acetylglucosamine (UDP-GlcNAc) at 2.2 Å and 3.1 Å resolution (termed Complex I and Complex II, respectively) (5). These structures revealed that CtLpxD contains three nearly identical protein chains within its asymmetric unit (5), which coincide with the three subunits

Xylella fastidiosa was the first plant pathogen to have its complete genome sequence elucidated. Routine database analyses suggested that two enzymes essential for fatty acid synthesis were missing, one of these is the holo-acyl-carrier-protein synthase. However, here we demonstrate, using 13 C NMR spectroscopy, that X. fastidiosa is indeed able to synthesize fatty acids from acetate via an apparently conventional metabolic pathway. We further identify a gene product HetI, an alternative phosphopantetheinyl transferase, which we propose to fill the missing link. Homology modeling of HetI shows conservation of the Coenzyme A binding site suggesting it to be an active enzyme and reveals several interesting structural features when compared with the surfactin synthaseactivating enzyme, on which the model was built. These include a simplified topology due to N-and C-terminal deletions and the observation of a novel serine ladder.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb’s cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP in...

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable success in the clinic, the biosynthetic prowess of these enzymes has been stymied by: 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. Towards filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KSCLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer. Using E. coli as a heterologous host, we obtained soluble proteins in titers representing significant improvements over previous KSCLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKSCLF indicates that these enzymes do not interact ...

The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4′-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB−) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2−, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial ...

Background: DynE8 is an iterative polyketide synthase (PKS) that assembles polyketide intermediates from acetate units derived from malonyl-CoA. Results: We report the first acyltransferase (AT DYN10) crystal structure for an iterative PKS. Conclusion: AT DYN10 protects the malonyl-enzyme, but not the acetyl-enzyme intermediate, from hydrolysis and facilitates the transfer of malonyl to the acyl carrier protein. Significance: This differs from the dual specificity exhibited by acyltransferases of mammalian FAS and other iterative PKSs. Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT DYN10) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a ␣/␤ hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a ␣/␤ fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude ␣-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser 651 residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates. * This work is supported by the Ministry of Education of Singapore through Academic Research Council grants (to Z.-X. L.), Biomedical Research Council grant 08/1/22/19/589, and an Action Thematique et Incitative sur Programme from the CNRS (to J. L.). □ S This article contains supplemental Tables S1 and Figs. S1-S6. The atomic coordinates and structure factors (codes 4AMP and 4AMM) have been deposited in the Protein Data Bank,

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activit relationships. Expression of the yeast NMT1 gene in Escherchia cofi, a bacterium that has no

The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7. Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD. The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein. TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7. In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7. The combination of L29 and ACP also stimulates Tn7 transposition in vitro. Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC⍣D reactions.

Serine palmitoyltransferase (SPT) catalyzes the first and committed step in sphingolipid biosynthesis condensating L-serine and acyl-CoA to form 3-oxo-sphinganine. Whenever the structural gene for SPT is present in genomes of Rhodobacteria (α-, β-, and γ-Proteobacteria), it co-occurs with genes coding for a putative acyl carrier protein (ACP) and a putative acyl-CoA synthetase (ACS). In the α-proteobacterium Caulobacter crescentus, CC_1162 encodes an SPT, whereas CC_1163 and CC_1165 encode the putative ACP and ACS, respectively, and all three genes are known to be required for the formation of the sphingolipid intermediate 3-oxo-sphinganine. Here we show that the putative ACP possesses a 4'-phosphopantetheine prosthetic group, is selectively acylated by the putative ACS and therefore is a specialized ACP (AcpR) required for sphingolipid biosynthesis in Rhodobacteria. The putative ACS is unable to acylate coenzyme A or housekeeping ACPs, but acylates specifically AcpR. Therefore,...

Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. InSinorhizobium meliloti1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence ofS. meliloti1021 also reveals the ORF SMc01553, annotated as a putative ACP.smc01553is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed inEscherichia coliwas labelledin vivowith [3H]β-alanine, a biosynthetic building block of the 4′-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4′-phosphopantetheine in the presence ofS. melilotiholo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase fromS....

Acyl carrier protein (ACP) of Escherichia coli is a small acidic protein which functions as carrier of growing acyl chains during their biosynthesis and as donor of acyl chains during transfer to target molecules. This unique ACP of E. coli is expressed constitutively. In more complex bacteria, multiple ACPs are present, indicating a channeling of pools of multi-carbon units into different biosynthetic routes. In rhizobia, for example, besides the constitutive ACP (AcpP) involved in the biosynthesis and transfer of common fatty acids, three specialized ACPs have been reported: (1) the flavonoid-inducible nodulation protein NodF, (2) AcpXL that transfers 27hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis, and (3) the RkpF protein which is required for the biosynthesis of rhizobial capsular polysaccharides. All three of those specialized rhizobial ACPs are required for the biosynthesis of cellsurface molecules that play a role in establishing the symbiotic relationship between rhizobia and their legume hosts. Surprisingly, the recently sequenced genomes from Mesorhizobium loti and Sinorhizobium meliloti suggest even more candidates for ACPs in rhizobia. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.