Crystal structure of the priming beta-ketosynthase from the R1128 polyketide biosynthetic pathway

ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.     

The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units

Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.     

Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9-C14 first-ring cyclization. However, how C7-C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 ? crystal structure of ZhuI, a C7-C12-specific first-ring ARO/CYC from the type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition that may be important for directing C7-C12 (rather than C9-C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the didomain ARO/CYCs from KR-containing type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7-C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.     

Kinetic analysis of the actinorhodin aromatic polyketide synthase.

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.     

In vitro synthesis of curcuminoids by type III polyketide synthase from Oryza sativa

Curcuminoids, major components of the spice turmeric, are used as a traditional Asian medicine and a food additive. Curcumin, a representative curcuminoid, has received a great deal of attention because of its anti-inflammatory, anticarcinogenic, and antitumor activities. Here we report a novel type III polyketide synthase named curcuminoid synthase from Oryza sativa, which synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA. The reaction begins with the thioesterification of the thiol moiety of Cys-174 by a starter molecule, 4-coumaroyl-CoA. Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate. Subsequent hydrolysis of the intermediate yields a beta-keto acid, which in turn acts as the second extender substrate. The beta-keto acid is then joined to the Cys-174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin. This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a beta-keto acid that subsequently serves as the second extender to form curcuminoids. Curcuminoid synthase appears to be capable of the synthesis of not only diarylheptanoids but also gingerol analogues, because it synthesized cinnamoyl(hexanoyl)methane, a putative intermediate of gingerol, from cinnamoyl-CoA and 3-oxo-octanoic acid.     

In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin

Monogalactosyldiacylglycerol (MGDG), a major membrane lipid of chloroplasts, is synthesized by MGDG synthase (MGD) localized in chloroplast envelope membranes. We investigated whether MGD activity is regulated in a redox-dependent manner using recombinant cucumber MGD overexpressed in Escherichia coli. We found that MGD activity is reversibly regulated by reduction and oxidation in vitro and that an intramolecular disulfide bond(s) is involved in MGD activation. Because thioredoxin efficiently reduced disulfide bonds to enhance MGD activity in vitro, MGD is potentially an envelope-bound thioredoxin target protein in higher plants.     

In silico Targeting,inhibition and analysis of polyketide synthase enzyme in Aspergillus ssp

Aflatoxins are toxic and carcinogenic components produced by some Aspergillus species such as Aspergillus flavus. Polyketide synthases enzyme (PKS) plays a central role in aflatoxin s biosynthesis of in Aspergillus flavus, especially the product template (PT) domain, which controls the aldol cyclization of the polyketide forerunner during the biosynthesis of the aflatoxin pathway process. Here, we apply the in silico approaches to validate 623 natural components obtained from the South African Natural Compound Database (SANCDB), to distinguish the PT domain s prospected inhibitors. From the 623 compounds, docking results showed that there are 330 different compounds with energy binding lower than the natural substrate (palmitic acid or PLM) of the Product Templet domain (PT). Three factors were selected to determine the best 10 inhibiting components; 1) energy binding, 2) the strengthen chemical interactions, 3) the drug-likeness. The top ten inhibiting components are kraussianone 6, kraussianone 1, neodiospyrin, clionamine D, bromotopsentin, isodiospyrin, spongotine A, kraussianone 3, 14β-Hydroxybufa-3,5,20,22-tetraenolide and kraussianone 7. The chemical interactions between 3HRQ domain and the natural substrate in the active site amino acids are highly similar to the 3HRQ with the top ten components, but the main differences are in the binding energy which is the best in the top ten ligands. Those ten components give successful inhibition with PT domain which will lead to the formula to be used for inhibition and control aflatoxin contamination of agriculture crop yields and lessen the degree of harming and sicknesses that are coming about because of acquiring measures of aflatoxin.     

In vitro analysis of the chain termination reaction in the synthesis of poly-(R)-beta-hydroxybutyrate by the class III synthase from Allochromatium vinosum

Allochromatium vinosum polyhydroxyalkanoate synthase catalyzes formation of poly-(R)-3-hydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-coenzyme A (HB-CoA). (R)-3-Hydroxybutyryl-N-acetylcysteamine (HB-NAC) is an alternative substrate for this synthase in vitro, with a turnover 1% that of HB-CoA. With HB-NAC, the molecular weight (M(w)) of PHB produced at substrate-to-enzyme ratios of 1500-15 000 is approximately 75 kDa. (1)H NMR shows that PHB produced has hydroxybutyrate at the alcohol end and N-acetylcysteamine (NAC) at the carboxylate end of the polymer. Exogenous NAC has no effect on the M(w) of PHB produced with HB-CoA or HB-NAC in vitro, whereas PHB from a polymerization reaction with both HB-CoA and HB-NAC has intermediate M(w). These results can be accommodated by two models. In the first, NAC liberated at the active site during polymerization acts as a chain transfer agent. In the second, there is a noncovalent polymer intermediate covalently linked to NAC, which can dissociate from the active site.     

In vitro reconstitution of demolybdosulfite oxidase by molybdate.

Reconstitution of purified demolybdosulfite oxidase from rat liver has been achieved using inorganic molybdate as the source of molybdenum. The activation process has a pH optimum of 7.4 and is dependent on concentrations of molybdate and demolybdoenzyme. The reaction is inhibited by high concentrations of anions and by reduction of the demolybdoenzyme and requires incubation temperatures higher than 30 degrees. A reconstitution mechanism involving loss of tungsten and concomitant replacement with molybdenum in those demolybdo molecules which contain tungsten is supported by the following observations: (a) the extent of activation achieved by molybdate corresponds to the proportion of molecules in the preparation which contain tungsten. (b) Incubation of the demolybdoenzyme preparation at 37 degrees in the absence of molybdate results in progressive and concentration-dependent loss of ability to be reconstituted by molybdate and a corresponding but more rapid loss of tungsten from the enzyme. The reconstituted enzyme displays the molybdenum EPR signal characteristic of native enzyme and is inactivated by incubation at 42 degrees in a manner identical to native sulfite oxidase.     

In vitro reconstitution of hnRNP particles

The assembly of hnRNP-like particles was studied by in vitro reconstitution, UV-crosslinking and CsCl-equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of p = 1.4 g/cm3 as 'native' 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the Cl-core hnRNP protein could be identified.     

In vitro reconstitution of pancreatic islets

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.     

In vitro reconstitution of pancreatic islets

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.     

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

In vitro reconstitution of the end replication problem

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.     

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.     

A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin.

Race T of the fungal pathogen Cochliobolus heterostrophus is highly virulent toward Texas male sterile (T) maize and differs from its relative, race O, at a locus (Tox1) that is responsible for the production of T-toxin, a family of linear long-chain (C35 to E41) polyketides. In a previous study, the restriction enzyme-mediated integration procedure was used to mutagenize and tag Tox1. Here, we report that the DNA recovered from the insertion site of one mutant encodes a 7.6-kb open reading frame (2530 amino acids) that identifies a multifunctional polyketide synthase (PKS)-encoding gene (PKS1) with six catalytic domains arranged in the following order, starting at the N terminus: beta-ketoacyl synthase, acyltransferase, dehydratase, enoyl reductase, beta-ketoacyl reductase, and acyl carrier protein. PKS1 is interrupted by four apparent introns (74, 57, 49, and 41 bp) and exists in the genome as a single copy surrounded by highly repetitive, A + T-rich DNA. When PKS1 in race T was inactivated by targeted gene disruption, T-toxin production and high virulence were eliminated, indicating that this PKS is required for fungal virulence. Race O strains, which do not produce T-toxin, lack a detectable homolog of PKS1, suggesting that race T may have acquired PKS1 by horizontal transfer of DNA rather than by vertical inheritance from an ancestral strain.     

In vitro reconstitution of the interactions in the PIDDosome

Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.     

In vitro reconstitution of wool intermediate filaments

Although the hard α-keratins of wool are recognized as members of the intermediate filaments by sequence comparison thus for all attempts on reconstitution of wool α-keratin in filaments in vitro have failed. Here we show the oxidative sulphitolysis rather than the previously used S-carboxymethylation is the method of choice to prepare α-keratin derivatives suitable for assembly experiments. Once the protecting S-sulpho group is removed by 2-mercaptoethanol in vitro filaments formation can be induced. Electron micrographs show filaments with a diameter of 7–11 nm as in all other intermediate filaments. Thus, filament formation of α-keratins does not require the presence of matrix proteins.