Common to all FASs, PKSs and NRPSs is a remarkable component, the acyl or peptidyl carrier protein (A/PCP). These take the form of small individual proteins in type II systems or discrete folded domains in the multi-domain type I systems and are characterized by a fold consisting of three major a-helices and between 60-100 amino acids. This protein is central to these biosynthetic systems and it must bind and transport a wide variety of functionalized ligands as well as mediate numerous protein-protein interactions, all of which contribute to efficient enzyme turnover. This review covers the structural and biochemical characterization of carrier proteins, as well as assessing their interactions with different ligands, and other synthase components. Finally, their role as an emerging tool in biotechnology is discussed.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.

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Three-coordinate bipyridyl complexes of gold, [(κ-bipy)Au(η-CH)][NTf], are readily accessed by direct reaction of 2,2'-bipyridine (bipy), or its derivatives, with the homoleptic gold ethylene complex [Au(CH)][NTf]. The cheap and readily available bipyridyl ligands facilitate oxidative addition of aryl iodides to the Au(I) center to give [(κ-bipy)Au(Ar)I][NTf], which undergo first aryl-zinc transmetalation and second C-C reductive elimination to produce biaryl products. The products of each distinct step have been characterized. Computational techniques are used to probe the mechanism of the oxidative addition step, offering insight into both the origin of the reversibility of this process and the observation that electron-rich aryl iodides add faster than electron-poor substrates. Thus, for the first time, all steps that are characteristic of a conventional intermolecular Pd(0)-catalyzed biaryl synthesis are demonstrated from a common monometallic Au complex and in the absence of directing groups.

The synthases that produce fatty acids in mammals (FASs) are arranged as large multidomain polypeptides. The growing fatty acid chain is bound covalently during chain elongation and reduction to the acyl carrier protein (ACP) domain that is then able to access each catalytic site. In this work we report the highresolution nuclear magnetic resonance (NMR) solution structure of the isolated rat fatty acid synthase apoACP domain. Fatty acid biosynthesis in mammals is important not only for energy homeostasis and development but also as a potential target for the treatment of obesity (1) and cancer (2). Type I fatty acid synthases (FASs) 3 that catalyze the biosynthesis of fatty acids in mammals, utilize simple acyl units, bound to the phosphopantetheine arm of a holo acyl carrier protein (ACP) domain, for chain initiation and elongation. The recent elucidation of the low resolution structure of the mammalian FAS by x-ray crystallography (3) has provided key structural and mechanistic insights into this important enzyme. Although the resolution of the crystal is insufficient to discern the backbone and side chains, the electron density has permitted the authors to propose a model based on the structures of individual domains and homologous enzymes. The authors have proposed a "head to head" dimeric model that comprises a central core consisting of the enol-reductase, dehydratase (DH), and ketosynthase with the malonyl transferase and ketoreductase domains being located peripherally. Dimerization occurs through association of the KS domains. Notable absences in the crystal structure are the locations of the peripheral ACP and thioesterase domains, suggesting that the positions of the ACP and thioesterase domains are relatively mobile compared with the core of the FAS. In comparison, bacterial Type II FASs consist of discrete monofunctional proteins (4). Structural studies of the Type II FAS ACP components are particularly well developed and have revealed the structural basis of acyl chain binding and the phenomenon of conformational switching. The crystal structures of Escherichia coli FAS butyryl (5), hexanoyl-, heptanoyl-, and decanoyl-ACPs (6) and nuclear magnetic resonance (NMR) structures of spinach FAS decanoyl-and stearoyl-ACPs have been reported (7). These structures reveal that during fatty acid biosynthesis, fully saturated acyl chains are sequestered within a central cavity in the ACP formed through conformational changes in the protein. The fatty acid chain is sequestered within the hydrophobic core of the ACP perhaps to protect the thioester moiety from hydrolysis. Binding of the acyl chain also influences the dynamics of the ACPs. Spinach FAS holo-ACP exists in equilibrium between a folded and largely disordered form, however, upon acylation this equilibrium is shifted toward the folded form. At present the physiological role of switching of this, and other ACPs, is unknown (8, 9). It has been suggested that switching confers allosteric regulation of the ACP, whereby its interaction with other enzymes...

Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.

In the actinorhodin type II polyketide synthase, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity (Javidpour, et al., 2011). The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified which are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides.