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.     

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.     

Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB.

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.     

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.     

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.     

Identification and substrate specificity of beta -ketoacyl (acyl carrier protein) synthase III (mtFabH) from Mycobacterium tuberculosis

The long-chain alpha-alkyl-beta-hydroxy fatty acids, termed mycolic acids, which are characteristic components of the mycobacterial cell wall are produced by successive rounds of elongation catalyzed by a multifunctional (type I) fatty acid synthase complex followed by a dissociated (type II) fatty acid synthase. In bacterial type II systems, the first initiation step in elongation is the condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) catalyzed by beta-ketoacyl-ACP III (FabH). An open reading frame in the Mycobacterium tuberculosis genome (Rv0533c), now termed mtfabH, was 37.3% identical to Escherichia coli ecFabH and contained the Cys-His-Asn catalytic triad signature. However, the purified recombinant mtFabH clearly preferred long-chain acyl-CoA substrates rather than acyl-ACP primers and did not utilize acetyl-CoA as a primer in comparison to ecFabH. In addition, purified mtFabH was sensitive to thiolactomycin and resistant to cerulenin in an in vitro assay. However, mtFabH overexpression in Mycobacterium bovis BCG did not confer thiolactomycin resistance, suggesting that mtFabH may not be the primary target of thiolactomycin inhibition in vivo and led to several changes in the lipid composition of the bacilli. The data presented is consistent with a role for mtFabH as the pivotal link between the type I and type II fatty acid elongation systems in M. tuberculosis. This study opens up new avenues for the development of selective and novel anti-mycobacterial agents targeted against mtFabH.     

Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12.

Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene- 4-thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional beta-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabB15(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented the fabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM-resistant fatty acid biosynthesis in vivo and in vitro. These data show that beta-ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance.     

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

Identification, substrate specificity, and inhibition of the Streptococcus pneumoniae beta-ketoacyl-acyl carrier protein synthase III (FabH)

In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

Streptomyces coelicolor RedP and FabH enzymes, initiating undecylprodiginine and fatty acid biosynthesis, exhibit distinct acyl-CoA and malonyl-acyl carrier protein substrate specificities

RedP is proposed to initiate undecylprodiginine biosynthesis in Streptomyces coelicolor by condensing an acyl-CoA with malonyl-ACP and is homologous to FabH that catalyzes the same reaction for initiation of fatty acid biosynthesis. Herein, we report the substrate specificities of RedP and FabH from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC). RedP activity was observed only with a pairing of acetyl-CoA and malonyl-RedQ, consistent with its proposed role in initiating the formation of acetyl-CoA-derived prodiginines. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. FabH demonstrated greater catalytic efficiency for isobutyryl-CoA in comparison with acetyl-CoA using malonyl-FabC, consistent with the observation that in streptomycetes, a broad mixture of fatty acids is synthesized, with those derived from branched-chain acyl-CoA starter units predominating. Diminished FabH activity was also observed using malonyl-RedQ with the same preference for isobutyryl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can produce branched-chain alkyl prodiginines.     

β-Ketoacyl-acyl carrier protein synthase III from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): properties,inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.     

Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.     

Acetylene-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.     

Engineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III

The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D(3) acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 microM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.     

Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-(4'-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thiolactomycin.     

Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase

The small genome of the Gram-positive bacterium Lactococcus lactis ssp. lactis IL1403 contains two genes that encode proteins annotated as homologues of Escherichia coli beta-hydroxyacyl-acyl carrier protein (ACP) reductase. E. coli fabG encodes beta-ketoacyl-acyl carrier protein (ACP) reductase, the enzyme responsible for the first reductive step of the fatty acid synthetic cycle. Both of the L. lactis genes are adjacent to (and predicted to be cotranscribed with) other genes that encode proteins having homology to known fatty acid synthetic enzymes. Such relationships have often been used to strengthen annotations based on sequence alignments. Annotation in the case of beta-ketoacyl-ACP reductase is particularly problematic because the protein is a member of a vast protein family, the short-chain alcohol dehydrogenase/reductase (SDR) family. The recent isolation of an E. coli fabG mutant strain encoding a conditionally active beta-ketoacyl-ACP reductase allowed physiological and biochemical testing of the putative L. lactishomologues. We report that expression of only one of the two L. lactis proteins (that annotated as FabG1) allows growth of the E. coli fabG strain under nonpermissive conditions and restores in vitro fatty acid synthetic ability to extracts of the mutant strain. Therefore, like E. coli, L. lactis has a single beta-ketoacyl-ACP reductase active with substrates of all fatty acid chain lengths. The second protein (annotated as FabG2), although inactive in fatty acid synthesis both in vivo and in vitro, was highly active in reduction of the model substrate, beta-ketobutyryl-CoA. As expected from work on the E. coli enzyme, the FabG1 beta-ketobutyryl-CoA reductase activity was inhibited by ACP (which blocks access to the active site) whereas the activity of FabG2 was unaffected by the presence of ACP. These results seem to be an example of a gene duplication event followed by divergence of one copy of the gene to encode a protein having a new function.     

Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action.

The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]acetate labeling of fatty acids under these conditions. In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis. By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth. Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures. Analysis of extracts of [35S]methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid. Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli thioesterase I or E. coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect. Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).