裂殖壶菌酰基载体蛋白(ACP)基因的克隆与表达

ACP (Acyl carrier protein, 酰基载体蛋白) 参与高度不饱和脂肪酸的PKS (Polyketide synthase) 生物合成途径。从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆。该序列开放读码框全长429 bp, 编码142个氨基酸, 等电点为5.04, 具有4′-磷酸泛酰巯基乙胺(4′-PP)的结合位点。利用BamHⅠ/HindⅢ双酶切, 并连接到原核表达载体pET-30a, 构建了pET-30a/acp表达载体, 转化宿主菌E.coli BL21(DE3), IPTG诱导表达。SDS-PAGE分析表明该蛋白得到高效表达。     

裂殖壶菌酰基载体蛋白(ACP)基因的克隆与表达

ACP(Acyl carrier protein,酰基载体蛋白)参与高度不饱和脂肪酸的PKS(Polyketide synthase)生物合成途径.从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆.该序列开放读码框全长429 bp,编码142个氨基酸,等电点为5.04,具有4'-磷酸泛酰巯基乙胺(4'-PP)的结合位点.利用BamH Ⅰ/Hind Ⅲ双酶切,并连接到原核表达载体pET-30a,构建了pET-30a/acp表达载体,转化宿主菌E.coll BL21(DE3),IPTG诱导表达.SDS-PAGE分析表明该蛋白得到高效表达.     

Noninvolvement of Acyl Carrier Protein with Citrate Synthase and Malate Synthase

Acyl carrier protein (ACP coli) was isolated from commercially grown Escherichia coli B and was acetylated by chemical methods. Biological activity of the synthesized acetyl-ACP coli was checked in an in vitro fatty acid-synthesizing system isolated from E. coli B. Since acetyl-ACP is preferred over acetyl-coenzyme A (CoA) as a substrate in these reactions, the possibility that it may substitute for acetyl-CoA in biosynthetically and oxidatively important cellular pathways (glyoxylate and Krebs cycles, respectively) was examined. Acetyl-ACP was tested for substrate activity with the enzyme of each cycle which has been found to utilize acetyl-CoA. Crystalline citrate synthase (EC 4.1.3.7) of porcine origin (Calbiochem) was found to be inactive with acetyl-ACP coli, which acted neither as a substrate nor as an inhibitor in the presence of acetyl-CoA. Malate synthase (EC 4.1.3.2) of the acetate type was isolated from acetate-grown cells of a mutant of E. coli K-12 (VGD(3)H(5)) and was also found to be inactive with acetyl-ACP coli. The significance of these results and of the recent discovery of another phospho-pantetheine-containing protein are discussed.     

Rapid in Vivo Acylation of Acyl Carrier Protein with Exogenous Fatty Acids in Spirodela oligorrhiza

Posttranslational acylation of several chloroplast proteins with palmitic acid was recently demonstrated in Spirodela oligorrhiza (AK Mattoo, M Edelman [1987] Proc Natl Acad Sci USA 84: 1497-1501). We have now identified an in vivo acylated, soluble protein having an apparent M_r of 10 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as an acylated form of acyl carrier protein (ACP). This 10-kilodalton protein is present in low abundance, and its acylation is light-stimulated. Turnover of the acyl moiety but not the apo-protein is rapid in the light. The acylated 10-kilodalton protein coelectrophoreses with in vitro synthesized palmitoyl-acyl carrier protein and is immunoprecipitated from soluble extracts with an antibody raised against spinach ACP. Cerulenin, an inhibitor of β-ketoacyl-ACP synthetase, inhibited in vivo acylation of Spirodela ACP. Cell-free extracts of Spirodela plants were able to catalyze the transfer of palmitate from palmitoyl-CoA to ACP, suggesting the existence in higher plants of a pathway for acylation of ACP that involves transacylation from acyl-CoA.     

Relations Between Coenzyme A and Presumptive Acyl Carrier Protein in Different Conditions of Streptococcal Growth

Exploration of the specific role of cystine in the postexponential growth of Streptococcus faecalis led to an inquiry into the fate of cellular coenzyme A (CoA) and acyl carrier protein (ACP), both of which depend for their biosynthesis on cystine and pantothenate as precursors. In S. faecalis cells labeled by growth in the presence of (14)C-pantothenate, the label could be separated on the basis of solubility at pH 2.1 into two fractions of sharply differing metabolic characteristics. The fractions were not purified, but the soluble (14)C behaved analytically like CoA, and the insoluble (14)C was considered to represent an ACP-like entity on the basis of circumstantial evidence. The fate of these two fractions under various conditions of growth was studied. When the medium contained an excess of the needed precursors, the cellular content of CoA and ACP appeared to remain constant during exponential growth, and in a molar ratio of about 4 CoA to 1 ACP. Cellular ACP, once formed, appeared to be stable under these conditions, but CoA was degraded and replaced at the rate of approximately 20% per division period. With restrictive levels of pantothenate in the medium, initially formed CoA disappeared during growth, as a result, apparently of being converted to ACP. However, when the resulting CoA-depleted cells were returned to a medium containing enough pantothenate, resumption of normal growth was preceded by a lag period, during which rapid conversion of ACP to CoA appeared to take place.     

Phosphopantethenylated Precursor Acyl Carrier Protein Is Imported into Spinach (Spinacia oleracea) Chloroplasts

Acyl carrier protein (ACP) is an essential cofactor of fatty acid synthase. In plants, ACP is synthesized in the cytosol as a larger precursor protein and then is imported into the plastid where it is processed to a smaller mature form. The active form of ACP uses a covalently linked 4[prime]-phosphopantetheine prosthetic group derived from coenzyme A to covalently bind the acyl intermediates during fatty acid synthesis. The prosthetic group is added to ACP by holoACP synthase. This enzyme activity is associated with both the plastidial subcellular fraction and the soluble, or cytoplasmic, fraction. To gain further insight into potential in vivo pathways for the synthesis and maturation of ACP, in this study we examined whether precursor holoACP can be imported by isolated spinach (Spinacia oleracea) chloroplasts. Precursor holoACP containing a [35S]phosphopantetheine prosthetic group was prepared, and the radiolabel was used to demonstrate import of the phosphopantethenylated protein into isolated chloroplasts. In addition, timed chloroplast import assays indicated that in vitro import of the phosphopantethenylated protein is at least as efficient as import of the precursor apoprotein. Evidence was also obtained for a low level turnover of the prosthetic group among endogenous plastidial ACPs when coenzyme A was supplied exogenously.     

海洋季也蒙毕赤酵母ACP基因的克隆及生物信息学分析

酰基载体蛋白是脂肪酸途径中重要的组件,能够结合脂肪酸代谢途径中各种脂肪酰基中间体,在脂肪酸代谢中是不可或缺的辅因子。以本实验室筛选保存的海洋季也蒙毕赤酵母基因组为模板,PCR扩增酰基载体蛋白基因,获得384 bp的目的片段。生物信息学分析显示,其具有完整的开放阅读框,编码127个氨基酸,有磷酸泛酰巯基乙胺结合位点,为非分泌型亲水性蛋白,不存在信号肽,存在9个潜在的磷酸化位点,二级结构和三级结构主要以α螺旋和无规则卷曲为主,与已知的酰基载体蛋白结构有很高的相似性。     

Rigidifying Acyl Carrier Protein Domain in Iterative Type I PKS CalE8 Does Not Affect Its Function

Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function.     

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe⁴⁵ in helix II and Phe¹⁸ in the α₁α₂ loop and a hydrogen bonding between Ser¹⁵ in helix I and Ile²⁰ in the α₁α₂ loop, resulting in its high thermal stability. Phe⁴⁵-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser⁵⁸ in the α₂α₃ loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.     

Change in Levels of Acyl Carrier Proteins in Greening Plants

The levels of acyl carrier proteins (ACP) in greening spinachcotyledons and greening oat leaves were examined by immunoblottingwith antiserum raised against spinach ACP I. Two isoforms ofACP, ACP I and ACP II, were found in spinach cotyledons, asthey were in the green leaves. The level of ACP II was higherthan that of ACP I in etiolated cotyledons. The level of ACPI increased markedly with greening. In the greened cotyledons,the major isoform was ACP I as was the case in green spinachleaves. In oat leaves, two isoforms were also identified, oatACPI (about 12kDa) and ACP II (about 17kDa), which cross-reactedwith the antiserum against spinach ACP I, but which were differentfrom spinach ACPs I and II. The levels of oat ACPs I and IIwere very low in etiolated leaves. The increase in levels ofboth ACPs corresponded to the change in the activity of fattyacid synthesis during illumination for 24 h. During furtherillumination for 24 h, the level of ACP II increased a littlein parallel with the change in the activity of fatty acid synthesis,whereas the level of ACP I increased somewhat more. The functionof oat ACPs I and II is discussed in connection with the formationof chloroplast. (Received March 27, 1989; Accepted September 18, 1989)     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.     

融合酰基载体蛋白可增强大肠杆菌重组蛋白的可溶性和热稳定性

目前,蛋白质可溶性和热稳定性已成为重组蛋白高效生产、功能应用和长久保存不可回避的问题,而使用酸性蛋白融合标签可能是其有效解决策略。酰基载体蛋白(ACP)是脂肪酸生物合成途径的必要组分,在大肠杆菌中为一个高度酸性的小分子多肽。将大肠杆菌ACP与几个热不稳定的靶蛋白[如小桐子抗坏血酸过氧化物酶1(Jc APX1)、大豆核酮糖-1,5-二磷酸羧化/加氧酶的活化酶2(GmRCA2)、大肠杆菌高丝氨酸O-转琥珀酰酶(EcMetA)]进行基因融合并使其在大肠杆菌中诱导表达,发现ACP融合能显著增强这些重组靶蛋白的可溶性。另外,对重组蛋白的热处理和酶活性分析发现,融合ACP还能极大提高这三个靶蛋白的热稳定性,并有效保护JcAPX1酶活免遭热失活,使其耐热性提高了至少2℃。ACP的这种效果推测可能与其高酸度特性有关,可以作为一个新的功能酸性融合标签。     

An NADH-Dependent Acetoacetyl-CoA Reductase from Euglena gracilis: Purification and Characterization, Including Inhibition by Acyl Carrier Protein

An NADH-dependent acetoacetyl-CoA reductase from Euglena gracilis variety bacillaris was extensively purified and characterized. Two different isoelectric forms of the reductase with identical characteristics otherwise were found. The reductase was noncompetitively inhibited by acyl carrier protein, K_i 5.6 micromolar at pH 5.4; this inhibition decreased with increasing pH or ionic strength. Coenzyme A was a competitive inhibitor, K_i 230 micromolar. Kinetic parameters with respect to acetoacetyl-CoA and NADH were sensitive to changes in pH and ionic strength.     

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.     

Down-regulation of Mitochondrial Acyl Carrier Protein in Mammalian Cells Compromises Protein Lipoylation and Respiratory Complex I and Results in Cell Death

The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.Eukaryotes employ two distinct systems for the synthesis of fatty acids de novo. The bulk of fatty acids destined for membrane biogenesis and energy storage are synthesized in the cytosolic compartment by megasynthases in which the component enzymes are covalently linked in very large polypeptides; this system is referred to as the type I fatty acid synthase (FAS)² (1, 2). A second system localized in mitochondria is composed of a suite of discrete, freestanding enzymes that closely resemble their counterparts in prokaryotes (3–10), which are characterized as type II FASs (11). Most of the constituent enzymes of the mitochondrial fatty acid biosynthetic system have been identified and characterized in fungi and animals; all are nuclear-encoded proteins that are transported to the matrix compartment of mitochondria. Fungi with deleted mitochondrial FAS genes fail to grow on non-fermentable carbon sources, have low levels of lipoic acid and elevated levels of mitochondrial lysophospholipids (12, 13). These observations indicate that the mitochondrial FAS may serve to provide the octanoyl precursor required for the biosynthesis of lipoyl moieties de novo, as well as providing fatty acids that are utilized in remodeling of mitochondrial membrane phospholipids (14). The mitochondrial FAS system in animals is less well characterized. However, kinetic analysis of the β-ketoacyl synthase enzyme responsible for catalysis of the chain extension reaction in human mitochondria suggested that this system is uniquely engineered to produce mainly octanoyl moieties and has limited ability to form long-chain products (9). Indeed, studies with a reconstituted system from bovine heart mitochondrial matrix extracts confirmed that octanoyl moieties are the main product and are utilized for the synthesis of lipoyl moieties (15). One of the key components of the prokaryotic and mitochondrial FAS systems is a small molecular mass, freestanding protein, the ACP, that shuttles substrates and pathway intermediates to each of the component enzymes. The mitochondrial ACP is localized primarily in the matrix compartment (16), but a small fraction is integrated into complex I of the electron transport chain (17–23). As is the case with many of the other 45 subunits of complex I, the role of the ACP subunit is unclear (24). To clarify the physiological importance of the mitochondrial FAS, and the mitochondrial ACP in particular, in mammalian mitochondrial function we have utilized an RNA interference strategy to knockdown the mitochondrial ACP in cultured HEK293T cells.     

CoMFA based de novo design of Pyrrolidine Carboxamides as Inhibitors of Enoyl Acyl Carrier Protein Reductase from Mycobacterium tuberculosis

InhA, the enoyl acyl carrier protein reductase (EACP reductase) from Mycobacterium tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective target for the development of anti-microbial agents. We report here, comparative molecular field analysis (CoMFA) studies and subsequent de novo ligand design using the LeapFrog program on pyrrolidine carboxamides, which have been reported as selective inhibitors of EACP reductase from Mycobacterium tuberculosis. The CoMFA model, constructed from the inhibitors used in this study has been successfully used to rationalize the structure-activity relationship of pyrrolidine carboxamides. The CoMFA model produced statistically significant results with cross-validated and conventional correlation coefficients of 0.626 and 0.953 respectively. Further, the predictive ability of CoMFA model was determined using a test set which gave predictive correlation coefficient r ² _pred of 0.880, indicating good predictive power. Finally, Leapfrog was used to propose 13 new pyrrolidine carboxamide analogues, based on the information derived from the CoMFA contour maps. The designed molecules showed better predicted activity using the CoMFA model with respect to the already reported systems; hence suggesting that newly proposed molecules in this series of compounds may be more potent and selective toward EACP reductase inhibition.