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.     

Specific inhibition of iturin biosynthesis by cerulenin

Cerulenin, an inhibitor of fatty acid synthesis, inhibits the biosynthesis of iturin by Bacillus subtilis. With a cerulenin concentration of 2 micrograms/mL, 50% inhibition was achieved. At this concentration, cerulenin does not affect growth or total protein synthesis but does inhibit the incorporation of sodium [14C]acetate, [14C]myristic acid, and [14C]asparagine into iturin. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, the inhibition of iturin and beta-amino acid syntheses by cerulenin is discussed in relation with lipid synthesis.     

Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi.

To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with [9,10-3H]myristic acid, and labeled proteins were analyzed by gel electrophoresis. Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V. harveyi ACP serum and comigration with acyl-ACP standards and [3H]beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels. Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total [3H]14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V. harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain. 14:0- and 12:0-ACP were also the predominant species labeled in complex medium. In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V. harveyi was incubated with [3H]acetate, indicating that acyl-ACP labeling with [3H]14:0 in vivo is not due to the total degradation of [3H]14:0 to [3H]acetyl coenzyme A followed by resynthesis. Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with [3H]beta-alanine fivefold, while total incorporation of [3H]14:0 was not affected, although a shift to shorter chain lengths was noted. Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography. The levels of incorporation of [3H] 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min. Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl donor.     

Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.     

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Only one of the five Ralstonia solanacearum long-chain 3-ketoacyl-acyl carrier protein synthase homologues functions in fatty acid synthesis

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C(16:0)), palmitoleic (C(16:1)) and cis-vaccenic (C(18:1)) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.     

Improved pinocembrin production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by engineering fatty acid synthesis

The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes β-ketoacyl-ACP synthase III (FabH) and β-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.     

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.     

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.     

A σW-dependent stress response in Bacillus subtilis that reduces membrane fluidity

Bacteria respond to physical and chemical stresses that affect the integrity of the cell wall and membrane by activating an intricate cell envelope stress response. The ability of cells to regulate the biophysical properties of the membrane by adjusting fatty acid composition is known as homeoviscous adaptation. Here, we identify a homeoviscous adaptation mechanism in Bacillus subtilis regulated by the extracytoplasmic function σ factor σ(W). Cell envelope active compounds, including detergents, activate a sense-oriented, σ(W)-dependent promoter within the first gene of the fabHa fabF operon. Activation leads to a decrease in the amount of FabHa coupled with an increase in FabF, the initiation and elongation condensing enzymes of fatty acid biosynthesis respectively. Downregulation of FabHa results in an increased reliance on the FabHb paralogue leading to a greater proportion of straight chain fatty acids in the membrane, and the upregulation of FabF increases the average fatty acid chain length. The net effect is to reduce membrane fluidity. The inactivation of the σ(W)-dependent promoter within fabHa increased sensitivity to detergents and to antimicrobial compounds produced by other Bacillus spp. Thus, the σ(W) stress response provides a mechanism to conditionally decrease membrane fluidity through the opposed regulation of FabHa and FabF.     

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).     

Feedback inhibition of fatty acid synthesis in tobacco suspension cells

The flux through many metabolic pathways is regulated through feedback inhibition on regulatory enzymes by endproducts of the pathway. Whether feedback inhibition occurs in fatty acid synthesis in plants has been investigated. The addition of exogenous oleic acid, in the form of oleoyl-Tween (Tween-18:1) caused a three- to fivefold decrease in the rate of [1-¹⁴C]acetate incorporation into tobacco suspension cell fatty acids. The decrease in acetate incorporation occurred rapidly upon addition of Tween-18:1 and appeared to be specific for fatty acid synthesis. In order to elucidate possible regulatory steps involved in the feedback regulation of fatty acid synthesis in plant cells, tobacco cell acyl-ACP intermediates were analyzed using a combination of [1-¹⁴C]acetate labeling and immunoblot analysis. Within 30 min of exogenous lipid addition, acetyl-ACP increased and long chain acyl-ACP decreased, whereas medium chain acyl-ACP levels remained constant. These acyl-ACP profiles observed during the feedback inhibition were those predicted to occur under conditions where the flux through fatty acid synthesis is decreased due to limiting levels of malonyl-CoA and therefore indicated that acetyl-CoA carboxylase (ACCase) was centrally involved in the feedback regulation of fatty acid synthesis. Immunoblot analysis showed that ACCase protein levels did not change during the feedback inhibition, indicating that the feedback inhibition of fatty acid synthesis in plant cells occurs through biochemical or post-translational modification of ACCase and possibly other fatty acid synthesis enzymes.     

哈氏弧菌脂酰-ACP合成酶的体外功能

【目的】系统鉴定哈氏弧菌脂酰-ACP合成酶(Acyl-ACP synthetase,Aas S)以不同链长游离脂肪酸和非脂肪链羧酸作为底物的体外催化反应。【方法】利用非变性蛋白质凝胶电泳和紫外分光光度计法从定性和定量两个方面分析了Aas S的体外催化功能与活性。【结果】Aas S能够催化不同链长直链的自由脂肪酸合成脂酰-ACP,其中以C6–C12作为底物时活性最高;以羟基脂肪酸作为底物的情况下,Aas S催化C8–C14的羟基脂肪酸有较高的活性。非脂肪链羧酸类作为底物的反应中,20种蛋白质氨基酸、苯甲酸和水杨酸均可以作为Aas S的底物,合成相应的脂酰-ACP。【结论】本研究系统地证明了哈氏弧菌脂酰-ACP合成酶(Aas S)对不同底物的不同催化活性,为生物体内氨基酸代谢和菌黄素合成代谢的研究提供了可行性的分析依据。     

Acyl-ACPs的规模化合成

脂酰-酰基载体蛋白(fatty acyl-acyl carrier protein, acyl-ACP)是多种生物合成途径中的酰基供体。因供给限制,体外研究常用类似物acyl-CoA替代,而CoA部分和ACP有较大差异,限制了相关酶对底物识别的认识。因此稳定获得大量acyl-ACP是体外研究相关酶的催化机制及其代谢途径的关键。研究以holo-ACP和C4~C18链长脂肪酸为底物,在哈氏弧菌acyl-ACP合成酶(Vibrio harveyi acyl-ACP synthetase, VhAasS)催化下合成不同碳链长度的acyl-ACP;通过高效液相色谱(HPLC)方法,确定不同碳链长度acyl-ACP的合成产率。结果表明:碳链为C4~C14的acyl-ACP产率均高于90.0%,16:0-ACP产率为85.9%,18:1-ACP产率仅为25.7%。通过加入Li ⁺优化反应体系,16:0-ACP、18:1-ACP的产率达90.0%。进一步优化扩大反应体系可稳定获得20mg以上acyl-ACP;最后,把合成的acyl-ACP应用到甘油-3-磷酸酰基转移酶催化的反应体系中。不同链长acyl-ACP的规模化合成研究,为体外研究相关酶的催化机制提供重要基础。     

Inhibition of unsaturated fatty acid synthesis in escherichia coli by the antibiotic cerulenin.

Low concentrations of cerulenin inhibit the growth of Escherichia coli by selectively blocking unsaturated fatty acid synthesis. This inhibition was relieved by unsaturated fatty acid supplements alone but not by saturated fatty acid supplements. The utilization of exogenous unsaturated fatty acids to sustain growth in the presence of cerulenin was confirmed by the analysis of bulk lipid composition. The effects of cerulenin on fatty acid synthesis were examined in vivo by pulse labeling with [14C]acetate and in vitro using [14C]malonyl-coenzyme A. In both cases, unsaturated fatty acid synthesis was inhibited by low concentrations of cerulenin with a stimulation of saturated fatty acid synthesis. Using mutant strains deficient in fatty acid synthesis, the effects of cerulenin on beta-ketoacyl-[acyl-carrier-protein] synthetases I and II were examined. Our results indicate that beta-ketoacyl-[acyl-carrier-protein] synthetase I is more sensitive to inhibition by cerulenin than beta-ketoacyl-[acyl-carrier-protein] synthetase II.     

Haemophilus influenzae Rd lacks a stringently conserved fatty acid biosynthetic enzyme and thermal control of membrane lipid composition

The organization of the fatty acid synthetic genes of Haemophilus influenzae Rd is remarkably similar to that of the paradigm organism, Escherichia coli K-12, except that no homologue of the E. coli fabF gene is present. This finding is unexpected, since fabF is very widely distributed among bacteria and is thought to be the generic 3-ketoacyl-acyl carrier protein (ACP) synthase active on long-chain-length substrates. However, H. influenzae Rd contains a homologue of the E. coli fabB gene, which encodes a 3-ketoacyl-ACP synthase required for unsaturated fatty acid synthesis, and it seemed possible that the H. influenzae FabB homologue might have acquired the functions of FabF. E. coli mutants lacking fabF function are unable to regulate the compositions of membrane phospholipids in response to growth temperature. We report in vivo evidence that the enzyme encoded by the H. influenzae fabB gene has properties essentially identical to those of E. coli FabB and lacks FabF activity. Therefore, H. influenzae grows without FabF function. Moreover, as predicted from studies of the E. coli fabF mutants, H. influenzae is unable to change the fatty acid compositions of its membrane phospholipids with growth temperature. We also demonstrate that the fabB gene of Vibrio cholerae El Tor N16961 does not contain a frameshift mutation as was previously reported.     

天蓝色链霉菌中长链3-酮脂酰ACP合成酶的功能

【背景】链霉菌属于放线菌科,在土壤环境中广泛分布。链霉菌具有复杂的形态分化和多样性的次生代谢网络,能产生大量具有生物活性的次级代谢产物,被广泛深入研究。【目的】天蓝色链霉菌是链霉菌的模式菌株,其脂肪酸合成代谢与次级代谢联系紧密,但目前脂肪酸合成代谢途径还不清楚,其长链3-酮脂酰ACP合成酶还未见报道。【方法】利用大肠杆菌FabF序列进行同源比对,发现天蓝色链霉菌A3(2)的基因组中,SCO2390(ScoFabF1)、SCO1266(ScoFabF2)、SCO0548(ScoFabF3)和SCO5886 (ScoRedR)具有较高的相似性,并具有保守的Cys-His-His催化活性中心,可能具有长链3-酮脂酰ACP合成酶活性。采用PCR扩增方法分别获得以上基因,连入表达载体pBAD24M后分别互补大肠杆菌fabB(ts)突变株和fabB(ts)fabF双突变株,并检测转化子的生长情况。以上基因与pET-28b连接后,在大肠杆菌BL21(DE3)中表达,并利用Ni-NTA纯化获得蛋白,体外测定其催化活性。将以上基因分别互补大肠杆菌fabF突变株后,GC-MS测定互补株的脂肪酸组成。【结果】4个同源基因中,只有ScofabF1能恢复fabB(ts)fabF双突变株42°C时在添加油酸条件下的生长,其他3个基因均不能恢复生长。而这4个基因都不能恢复fabB(ts)突变株42°C时生长。体外活性测定ScoFabF1具有长链3-酮脂酰ACP合成酶活性,其他3个蛋白都不具有该活性。仅ScofabF1能显著提高大肠杆菌fabF突变株的顺-11-十八碳烯酸(C18:1)比例,其他3个基因都不具有该功能。【结论】天蓝色链霉菌中ScofabF1编码长链3-酮脂酰ACP合成酶II,在脂肪酸利用过程中发挥重要作用。天蓝色链霉菌中没有发现编码长链3-酮脂酰ACP合成酶I的基因,其可能通过其他途径合成少量的不饱和脂肪酸。以上研究结果为进一步研究天蓝色链霉菌中脂肪酸合成机制奠定了基础。