Evidence for a protein-protein interaction motif on an acyl carrier protein domain from a modular polyketide synthase

During biosynthesis on modular polyketide synthases (PKSs), chain extension intermediates are tethered to acyl carrier protein (ACP) domains through phosphopantetheinyl prosthetic groups. Each ACP must therefore interact with every other domain within the module, and also with a downstream acceptor domain. The nature of these interactions is key to our understanding of the topology and operation of these multienzymes. Sequence analysis and homology modeling implicates a potential helical region (helix II) on the ACPs as a protein-protein interaction motif. Using site-directed mutagenesis, we show that residues along this putative helix lie at the interface between the ACP and the phosphopantetheinyl transferase that catalyzes its activation. Our results accord with previous studies of discrete ACP proteins from fatty acid and aromatic polyketide biosynthesis, suggesting that helix II may also serve as a universal interaction motif in modular PKSs.     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.     

Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

Production of vancomycin aglycone conjugated to a peptide carrier domain derived from a biosynthetic non-ribosomal peptide synthetase

A method for attaching the vancomycin aglycone to a peptide carrier domain (PCD) is reported which involves reacting the apo-PCD produced in Escherichia coli with vancomycin aglycone-coenzyme A thioester, catalyzed by the phosphopantetheinyl transferase Sfp from Bacillus subtilis.     

The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs

While X-ray and NMR structures are now available for most components of the Type II fatty acid synthase (FAS), there are no structures for Type I FAS domains. A region from the mammalian (rat) FAS, including the putative acyl carrier protein (ACP), has been cloned and over-expressed. Here we report multinuclear, multidimensional NMR studies which show that this isolated ACP domain contains four alpha-helices (residues 8-16 [1]; 41-51 [2]; 58-63 [3] and 66-74 [4]) and an overall global fold characteristic of ACPs from both Type II FAS and polyketide synthases (PKSs). The overall length of the structured ACP domain (67 residues) is smaller than the structured regions of the Eschericia coli FAS ACP (75 residues), the actinorhodin PKS ACP (78 residues) and the Bacillus subtilis FAS ACP (76 residues). We further show that the rat FAS ACP is recognised as an efficient substrate by enzymes known to modify Type II ACPs including phosphopantetheinyl and malonyl transferases, but not by the heterologous S. coelicolor minimal polyketide synthase.     

Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 ? germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.     

Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis.

BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known. RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process. CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules

Many polyketides are synthesized by a class of multifunctional enzymes called type I modular polyketide synthases (PKSs). Several reports have described the power of predictively altering polyketide structure by replacing individual PKS domains with homologues from other PKSs. For example, numerous erythromycin analogues have been generated by replacing individual methylmalonyl-specific acyl transferase (AT) domains of the 6-deoxyerythronolide B synthase (DEBS) with malonyl-, ethylmalonyl-, or methoxymalonyl-specific domains. However, the construction of hybrid PKS modules often attenuates product formation both kinetically and distributively. The molecular basis for this mechanistic imperfection is not understood. We have systematically analyzed the impact of replacing an AT domain of DEBS on acyl-AT formation, acyl-CoA:HS-NAc acyl transferase activity, acyl-CoA:ACP acyl transferase activity (nucleophile charging), acyl-SNAc:ketosynthase acyl transferase activity (electrophile charging), and beta-ketoacyl ACP synthase activity (condensation). As usual, domain junctions were located in interdomain regions flanking the AT domain. Kinetic analysis of hybrid modules containing either malonyl transferase or methylmalonyl transferase domains revealed a 15-20-fold decrease in overall turnover numbers of the hybrid modules as compared to the wild-type module. In contrast, both the activity and the specificity of the heterologous AT domains remained unaffected. Moreover, no defects could be detected in the ability of the heterologous AT domains to catalyze acyl-CoA:ACP acyl transfer. Single turnover studies aimed at directly probing the ketosynthase-catalyzed reaction led to two crucial findings. First, wild-type modules catalyzed chain elongation with comparable efficiency regardless of whether methylmalonyl-ACP or malonyl-ACP were the nucleophilic substrates. Second, chain elongation in all hybrid modules tested was seriously attenuated relative to the wild-type module. Our data suggest that, as currently practiced, the most deleterious impact of AT domain swapping is not on the substrate specificity. Rather, it is due to the impaired ability of the KS and ACP domains in the hybrid module to catalyze chain elongation. Consistent with this proposal, limited proteolysis of wild-type and hybrid modules showed major differences in cleavage patterns, especially in the region between the KR and ACP domains.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

First in vitro directed biosynthesis of new compounds by a minimal type II polyketide synthase: evidence for the mechanism of chain length determination

The minimal actinorhodin polyketide synthase bearing two point mutations (KSbeta Q161A, ACP C17S) was chemically modified to carry novel C4 to C8 starter units on the ACP: on incubation with an excess of malonyl CoA new 16-carbon polyketides are made, supporting a measuring mechanism.     

Protein assembly line components in prodigiosin biosynthesis: characterization of PigA,G,H,I,J

The red streptomycete metabolite prodigiosin has a unique tripyrrolic structure with two of the three pyrrolyl moieties in tandem. Five enzymes, PigA,G,H,I, and J, are involved in dipyrrole (rings A and B) formation. We have heterologously expressed and purified from Escherichia coli these five enzymes. At first, pyrrole ring A is formed on the peptidyl carrier protein PigG by one of two possible ways: (i) by action of the adenylation domain PigI that transforms l-proline into l-prolyl-AMP and by the flavoprotein dehydrogenase PigA responsible for the four-electron oxidation reaction; (ii) by loading with the pyrrolyl-2-carboxyl-(S)-pantetheinyl moiety from synthetic pyrrolyl-CoA using the phosphopantetheinyl transferase Sfp. Subsequently, pyrrole ring B is constructed by PigH after the transfer of ring A to the ketosynthase of PigJ. PigH consists of three domains: two acyl carrier proteins (ACPs) and a seryltransferase (SerT). Using HPLC and nanospray-Fourier Transform Mass Spectrometry (nFTMS), we established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.     

The malonyl transferase activity of type II polyketide synthase acyl carrier proteins

Acyl carrier proteins (ACPs) play a fundamental role in directing intermediates among the enzyme active sites of fatty acid and polyketide synthases (PKSs). In this paper, we demonstrate that the Streptomyces coelicolor (S. coelicolor) actinorhodin (act) PKS ACP can catalyze transfer of malonate to type II S. coelicolor fatty acid synthase (FAS) and other PKS ACPs in vitro. The reciprocal transfer from S. coelicolor FAS ACP to a PKS ACP was not observed. Several mutations in both act ACP and S. coelicolor FAS ACP could be classified by their participation in either donation or acceptance of this malonyl group. These mutations indicated that self-malonylation and malonyl transfer could be completely decoupled, implying that they were separate processes and that a FAS ACP could be converted from a non-malonyl-transferring protein to one with malonyl transferase activity.     

Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus

Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.     

胆盐与磷酸钙的相互作用

胆汁的pH条件下(pH=6～8)，应该生成无定形磷酸钙(ACP)，而在胆结石中磷酸钙通常以羟基磷灰石的形式出现.利用谱学方法研究了ACP与胆盐的作用.结果表明，胆盐以胶团的形式与ACP作用，在溶液中形成复合胶团，使其溶解度增加.不同类型胆盐与ACP的作用能力不同：脱氧胆酸钠(NaDC) > 牛磺胆酸钠(NaTC) > 胆酸钠(NaC).胆盐与ACP中结合钙的亲和能力大于结合钙的亲和能力，使ACP在胆汁的环境下容易转化为羟基磷灰石.     

Labeling proteins with small molecules by site-specific posttranslational modification

We report here the development of a general strategy for site-specific labeling of proteins with small molecules by posttranslational modification enzyme, phosphopantetheinyl transferase Sfp. The target proteins are expressed as fusions to the peptide carrier protein (PCP) excised from nonribosomal peptide synthetase, and Sfp catalyzes the covalent modification of a specific serine residue on PCP by the small molecule-phosphopantetheinyl conjugate. The labeling reaction proceeds with high specificity and efficiency, targeting PCP fusion proteins in the cell lysate. The PCP tag has been shown to be compatible with various proteins, and Sfp-catalyzed PCP modification, compatible with various small-molecule probes conjugated to coenzyme A, highlighting the potential of the PCP tag for site-specific protein labeling with small molecules.     

Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA:ACP transacylase

Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.     

Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains

Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates. To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase. A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP. With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates.     

Microwave‐Assisted Hydrothermal Rapid Synthesis of Amorphous Calcium Phosphate Mesoporous Microspheres Using Adenosine 5′‐Diphosphate and Application in pH‐Responsive Drug Delivery

Herein we report a rapid and green strategy for the preparation of amorphous calcium phosphate mesoporous microspheres (ACP‐MSs) using adenosine 5′‐diphosphate disodium salt (ADP) as an organic phosphorus source by a microwave‐assisted hydrothermal method. The effects of the pH value, the reaction time, and temperature on the crystal phase and morphology of the product are investigated. The ADP biomolecules used in this strategy play an important role in the formation of ACP‐MSs. The as‐prepared ACP‐MSs are efficient for anticancer drug delivery by using doxorubicin (Dox) as a model drug, and the Dox‐loaded ACP‐MSs show a high ability to damage cancer cells. Moreover, the ACP‐MSs drug delivery system exhibits a pH‐responsive drug‐release behavior due to the degradation of ACP‐MSs at a low pH value, thus, it is promising for applications in pH‐responsive drug delivery.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.