Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep‐sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl‐reduction. In this work, we have used the Udwary‐Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA‐homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl‐CoA to β‐hydroxybutyryl‐CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA‐homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA‐like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA‐homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.     

Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA

A crude extract from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin was previously shown to incorporate methylmalonyl-CoA into mycocerosic acids, exemplified by 2,4,6,8-tetramethyloctacosanoic acid, and malonyl-CoA into n-fatty acids (Rainwater D. L., and Kolattukudy, P. E. (1983) J. Biol. Chem. 258, 2979-2985). The presence of several fatty acid synthases with differences in substrate preference and product chain length was detected in the crude extract of M. tuberculosis var. bovis. Among them was a mycocerosic acid synthase which was purified to homogeneity using anion-exchange chromatography, gel filtration, affinity chromatography, and hydroxylapatite chromatography. This fatty acid synthase elongated long-chain fatty acyl-CoA primers using methylmalonyl-CoA and NADPH to produce multimethyl-branched mycocerosic acids. The enzyme was specific for methylmalonyl-CoA and would not incorporate malonyl-CoA into fatty acids. It elongated n-C6 to n-C20 CoA esters to generate primarily the corresponding tetramethyl-branched mycocerosic acids. Exogenous [1-14C]acyl-CoA and trideuteromethylmalonyl-CoA were incorporated into the multimethyl-branched fatty acids. Dodecyl sulfate electrophoresis showed that the enzyme had a molecular weight of 238,000, whereas gel filtration showed a native molecular weight of 490,000, indicating that the enzyme is composed of two monomers of identical molecular weight. The enzyme contained an acyl carrier protein-like segment as indicated by incorporation of [1-14C] pantothenate into the 238-kDa protein and production of 1 mol of taurine/mol of the monomer upon hydrolysis of performic acid-oxidized enzyme. It is concluded that the mycocerosic acid synthase is a multifunctional enzyme similar to the well-characterized multifunctional fatty acid synthases except for the substrate specificity.     

Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.     

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.     

The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.     

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

Evolution of acyl-ACP thioesterases and β-ketoacyl-ACP synthases revealed by protein–protein interactions

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein–protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners, whereas KSs showed promiscuous behavior across bacterial, plant, and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic cross-linking, and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.     

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether, LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

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.     

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.     

Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG.

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.     

Fat metabolism in higher plants. Characterization of plant acyl-ACP and acyl-CoA hydrolases.

Avocado mesocarp extracts contain both acyl-CoA and acyl-acyl carrier protein (ACP) hydrolase activities. These activities have been separated by a sequence of ammonium sulfate fractionations, DEAE-cellulose, and hydroxyapatite column chromatography. Two distinct acyl-CoA hydrolase fractions and one acyl-ACP hydrolase fraction were obtained. The acyl-ACP hydrolase fraction was essentially free of acyl-CoA hydrolase activity, had a pH optimum of 9.5 and a molecular weight of 70–80,000 based on sucrose density gradient centrifugation and gel filtration chromatography. Substrate specificity studies showed that lauroyl-ACP, myristoyl-ACP, palmitoyl-ACP, and stearoyl-ACP were slowly hydrolyzed but oleoyl-ACP was rapidly hydrolyzed to free fatty acid. These results suggest a possible role for acyl-ACP hydrolase as one component of a switching system which allows, indirectly, acyl transfer from ACP to CoA derivatives in plant cells.     

Probing the mechanism of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These alpha-alkyl, beta-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C(24)-C(26) fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The beta-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg(46) revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg(161) --> Ala substitution. Our structural studies suggested that His(258), previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys(122).     

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.     

Obscurin is a semi‐flexible molecule in solution

Obscurin, a giant modular cytoskeletal protein, is comprised mostly of tandem immunoglobulin‐like (Ig‐like) domains. This architecture allows obscurin to connect distal targets within the cell. The linkers connecting the Ig domains are usually short (3–4 residues). The physical effect arising from these short linkers is not known; such linkers may lead to a stiff elongated molecule or, conversely, may lead to a more compact and dynamic structure. In an effort to better understand how linkers affect obscurin flexibility, and to better understand the physical underpinnings of this flexibility, here we study the structure and dynamics of four representative sets of dual obscurin Ig domains using experimental and computational techniques. We find in all cases tested that tandem obscurin Ig domains interact at the poles of each domain and tend to stay relatively extended in solution. NMR, SAXS, and MD simulations reveal that while tandem domains are elongated, they also bend and flex significantly. By applying this behavior to a simplified model, it becomes apparent obscurin can link targets more than 200 nm away. However, as targets get further apart, obscurin begins acting as a spring and requires progressively more energy to further elongate.     

A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases

Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies.     

共价交联捕获聚酮合成中的酮基还原酶和酰基载体蛋白结构域的瞬时复合物

【背景】在模块化聚酮合成酶(polyketidesynthase,PKS)的催化过程中,催化结构域与同源酰基载体蛋白(acyl-carrierprotein,ACP)之间的蛋白质-蛋白质相互作用起重要作用,但这种瞬时可逆的相互作用难以捕捉分析。【目的】获得ACP和酮基还原酶(ketoreductase,KR)相互作用的蛋白复合物。【方法】在KR和ACP之间的Linker上插入烟草蚀纹病毒(tobacco etch virus,TEV)蛋白酶切位点,通过双功能马来酰亚胺试剂BMH将KR和ACP共价交联,随后TEV酶切检测交联结果。调整反应条件,使交联效率最大化。根据KR-ACP交联复合物与体系内其他蛋白标签和分子量的差异,通过亲和层析和凝胶过滤等纯化手段,获得纯度较高的KR-ACP稳定交联复合物。【结果】单独表达的KR和ACP结构域交联不成功,融合表达的KR+ACP双结构域可以有效交联,结合使用亲和层析和凝胶过滤等纯化手段成功获得纯度较高的复合物。该策略可运用于多个KR和ACP的共价交联。【结论】建立了捕获并纯化KR和ACP瞬时相互作用复合物的有效方法,为后期晶体结构的解析、KR与ACP相互作用机理的揭示及参与相互作用关键氨基酸的鉴定提供了实验基础。