Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein.

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.     

Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12.

beta-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissociated (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region of the Escherichia coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymatic defect in fabH1 strains. The NruI-HindIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelectric point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the amino-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overproduction of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.     

Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea.

Localization of the enzymatic domains in the three multifunctional polypeptides from Saccharopolyspora erythraea involved in the formation of the polyketide portion of the macrolide antibiotic erythromycin was determined by computer-assisted analysis. Comparison of the six synthase units (SU) from the eryA genes with each other and with mono- and multifunctional fatty acid and polyketide synthases established the extent of each beta-ketoacyl acyl-carrier protein (ACP) synthase, acyltransferase, beta-ketoreductase, ACP, and thioesterase domain. The extent of the enoyl reductase (ER) domain was established by detecting similarity to other sequences in the database. A segment containing the putative dehydratase (DH) domain in EryAII, with a potential active-site histidine residue, was also found. The finding of conservation of a portion of the DH-ER interdomain region in the other five SU, which lack these two functions, suggests a possible evolutionary path for the generation of the six SU.     

Structure of RhlG, an essential beta-ketoacyl reductase in the rhamnolipid biosynthetic pathway of Pseudomonas aeruginosa

Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the beta-ketoacyl-acyl carrier protein (ACP) reductase of type II fatty acid synthesis. Crystallographic analysis reveals that the overall structures of the RhlG.NADP+ and FabG.NADP+ complexes are indeed similar, but there are key differences related to function. RhlG does not undergo the conformational changes upon NADP(H) binding at the active site that in FabG are the structural basis of negative allostery. Also, the acyl chain-binding pocket of RhlG is narrow and rigid compared with the larger, flexible substrate-binding subdomain in FabG. Finally, RhlG lacks a positively charged/hydrophobic surface feature adjacent to the active site that is found on enzymes like FabG that recognize the ACP of fatty acid synthesis. RhlG catalyzed the NADPH-dependent reduction of beta-ketodecanoyl-ACP to beta-d-hydroxydecanoyl-ACP. However, the enzyme was 2000-fold less active than FabG in carrying out the same reaction. These structural and biochemical studies establish RhlG as a NADPH-dependent beta-ketoacyl reductase of the SDR protein superfamily and further suggest that the ACP of fatty acid synthesis does not carry the substrates for RhlG.     

Effect of thiolactomycin on the individual enzymes of the fatty acid synthase system in Escherichia coli

Thiolactomycin, an antibiotic with the structure of (4S)-(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-++ +thiolide, selectively inhibits type II fatty acid synthases. The mode of the thiolactomycin action on the fatty acid synthase system of Escherichia coli was investigated. Of the six individual enzymes of the fatty acid synthase system, [acyl-carrier-protein] (ACP) acetyltransferase and 3-oxoacyl-ACP synthase were inhibited by thiolactomycin. On the other hand, the other enzymes were not affected by this antibiotic. The thiolactomycin inhibition of the fatty acid synthase system was reversible. As to ACP acetyltransferase, the inhibition was competitive with respect to ACP and uncompetitive with respect to acetyl-CoA. As to 3-oxoacyl-ACP synthase, the inhibition was competitive with respect to malonyl-ACP and noncompetitive with respect to acetyl-ACP. The thiolactomycin action on the fatty acid synthase system was compared with that of cerulenin.     

Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.     

Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria

The first condensation reaction in the fatty acid biosynthetic pathway in Escherichia coli was rate-limiting as judged by analysis of the relative pool sizes of acyl carrier protein (ACP) thioester intermediates in vivo. Comparable concentrations of acetyl-ACP, malonyl-ACP, and nonesterified ACP were present during logarithmic growth, whereas long-chain acyl-ACP comprised a minor fraction of the total ACP pool. The antibiotic cerulenin was used to irreversibly inhibit both beta-ketoacyl-ACP synthases I and II. However, acyl-ACP formation in vivo was not blocked by this antibiotic, and short-chain (4-8-carbon) acyl-ACPs increased to 60% of the total ACP pool in cerulenin-treated cells. These data suggested that existence of a cerulenin-resistant condensing enzyme that was capable of catalyzing the initial steps in chain elongation. A unique enzymatic activity, acetoacetyl-ACP synthase, that specifically catalyzed the condensation of malonyl-ACP and acetyl-ACP was detected in E. coli cell extracts. Acetoacetyl-ACP synthase activity was not inhibited by cerulenin and was present in extracts prepared from a double mutant harboring genetic lesions in beta-ketoacyl-ACP synthases I and II (fabB20 fabF3). These data point to the condensation of malonyl-ACP and acetyl-ACP as the rate-controlling reaction in fatty acid biosynthesis and implicate acetoacetyl-ACP synthase as the pacemaker of fatty acid production in organisms and organelles that possess dissociated (Type II) fatty acid synthase systems.     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.     

The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes.

The gene encoding Escherichia coli acyl carrier protein (ACP) has been isolated and sequenced. The ACP gene (called acpP) was located on the genetic map between fabF and fabD which encode two fatty acid biosynthetic enzymes, 3-ketoacyl-ACP synthase II and malonyl CoA-ACP transacylase, respectively. An open reading frame between acpP and fabD encodes a 26.5-kDa protein that has significant sequence identity (greater than 40%) with two acetoacetyl-CoA reductases and thus is believed to encode a 3-ketoacyl-ACP reductase. This gene (called fabG) is cotranscribed with acpP. Thus, the gene encoding ACP, the key carrier protein of fatty acid synthesis, is located within a cluster of fatty acid biosynthetic genes.     

大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白：FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.     

Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein.

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.     

Catalysis,specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.     

A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.     

Mitochondrial fatty acid synthesis in Trypanosoma brucei

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 beta-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 [(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.     

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.     

Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism

Bacteria respond to nutritional stresses by producing an intracellular alarmone, guanosine 5'-(tri)diphosphate, 3'-diphosphate [(p)ppGpp], which triggers the stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, upon fatty acid or carbon starvation, SpoT enzyme activity switches from (p)ppGpp degradation to (p)ppGpp synthesis, but the signal and mechanism for this response remain totally unknown. Here, we characterize for the first time a physical interaction between SpoT and acyl carrier protein (ACP) using affinity co-purifications and two-hybrid in E. coli. ACP, as a central cofactor in fatty acid synthesis, may be an ideal candidate as a mediator signalling starvation to SpoT. Accordingly, we show that the ACP/SpoT interaction is specific of SpoT and ACP functions because ACP does not interact with the homologous RelA protein and because SpoT does not interact with a non-functional ACP. Using truncated SpoT fusion proteins, we demonstrate further that ACP binds the central TGS domain of SpoT, consistent with a role in regulation. The behaviours of SpoT point mutants that do not interact with ACP reveal modifications of the balance between the two opposite SpoT catalytic activities thereby changing (p)ppGpp levels. More importantly, these mutants fail to trigger (p)ppGpp accumulation in response to fatty acid synthesis inhibition, supporting the hypothesis that the ACP/SpoT interaction may be involved in SpoT-dependent stress response. This leads us to propose a model in which ACP carries information describing the status of cellular fatty acid metabolism, which in turn can trigger the conformational switch in SpoT leading to (p)ppGpp accumulation.     

Functional expression of an acyl carrier protein (ACP) from Azospirillum brasilense alters fatty acid profiles in Escherichia coli and Brassica juncea.

Acyl carrier protein (ACP) is a central cofactor for de novo fatty acid synthesis, acyl chain modification and chain-length termination during lipid biosynthesis in living organisms. Although the structural and functional organization of the ACPs in bacteria and plant are highly conserved, the individual ACP is engaged in the generation of sets of signature fatty acids required for specific purpose in bacterial cells and plant tissues. Realizing the fact that the bacterial ACP being originated early in molecular evolution is characteristically different from the plant's counterpart, we explored the property of an ACP from Azospirillum brasilense (Ab), a plant-associative aerobic bacterium, to find its role in changing the fatty acid profile in heterologous systems. Functional expression of Ab-ACP in Escherichia coli, an enteric bacterium, and Brassica juncea, an oil-seed crop plant, altered the fatty acid composition having predominantly 18-carbon acyl pool, reflecting the intrinsic nature of the ACP from A. brasilense which usually has C18:1 rich membrane lipid. In transgenic Brassica the prime increment was found for C18:3 in leaves; and C18:1 and C8:2 in seeds. Interestingly, the seed oil quality of the transgenic Brassica potentially improved for edible purposes, particularly with respect to the enhancement in the ratio of monounsaturated (C18:1)/saturated fatty acids, increment in the ratio of linoleic (C18:2)/linolenic (C18:3) and reduction of erucic acid (C22:1).     

A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus

A heat-stable factor, required for de novo synthesis of fatty acids in the erythromycin-producing organism Streptomyces erythraeus, has been purified to homogeneity and identified as an acyl carrier protein (ACP). We conclude that, contrary to previous belief, fatty acid synthase in S. erythraeus more closely resembles the dissociable complex of E. coli than the tightly associated, multifunctional enzyme complex found in the related actinomycete Mycobacterium smegmatis.