Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers has been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins.

Structure-based design of synthetic peptide-based molecules that mimic the functional site of natural proteins, plays an important role in drug discovery nowadays. [1][2][3][4][5] Their application is widespread, ranging from synthetic antiviral, [3, 6] antifertility, [1,2,7] or antitumor [2,7, 8] agents to therapeutic agents that are able to mimic [9] or disrupt [4, 10] protein-protein interactions. A variety of structural mimics exist for a-helices, [11,12] b-turns or hairpins, [11, 13] and b-sheets. [11,14] However, more complex topologies, like four-helix bundles, [15] are often needed in order to mimic protein function adequately.[16] The total synthesis of such complex structures is generally demanding; this limits their application and emphasizes the need for high-efficiency synthetic strategies. In this communication, we describe a onestep procedure for the immobilization of (multiple) peptide loops onto a synthetic scaffold (Scheme 1) starting from a linear peptide. The reaction is extremely fast and clean and runs very well with linear peptides that are 2-30 amino acids long (> 30 not tested). It is compatible with all possible unprotected side-chain functionalities (except for free cysteine). It therefore avoids the need for complex synthetic strategies and this makes the reaction highly versatile with a very wide scope.As part of our research program on the mapping and reconstruction of the discontinuous epitope of follicle-stimulating hormone (FSH), [17] which is a heterodimeric member of the cysteine-knot protein family, [18] we recently discovered the fast and quantitative cyclization of dicysteine-containing peptides upon their treatment with a,a'-dibromoxylenes (T2). In organic solvents such as ACN, the reaction is rather slow and unselective, [19] but it becomes unusually fast and entirely selective for cysteines when performed in aqueous solutions.[20] For example, treatment of a 0.5 mm solution of the peptide *CRVPGDAHHADSLC# (1 a, where * = acetyl and # = amide) with 1.05 equiv of m-T2 in a 1:7 mixture of ACN/NH 4 HCO 3 (20 mm, pH 7.8) gives the corresponding monocyclic product 2 a with > 80 % yield in less than 15 min at RT (see Table 1). The corresponding intramolecular SS-dimer 3 is not formed (< 5 %) as oxidative cyclization is not competitive under these conditions. There is no doubt that the reaction takes place exclusively at the free sulfhydryl groups, since corresponding peptides without sulfhydryl groups [21] do not react at all with T2 scaffolds in the solvent system used.The difference in reactivity amongst various dicysteine-containing peptides that we have studied is negligible. The half-lives of peptides 1 a-g in the reaction with m-T2 vary only slightly (t 1/2 = 1.4-3.0 min, see Table 1), despite the fact that their length (14-42) and the number of amino acids that separate the two cysteines (0-22) are very different. In sharp contrast to this, there is a large difference in reactivity amongst different scaffolds. o-T2 (average t 1/2 = 1.4 min) is slightly more reactiv...

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases’ many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG). Thus, the diselenide bond of GSeSeG is 7 kcal/mol more stable than the disulfide bond of GSSG. Nonetheless, we found that GSeSeG can be used to oxidize cysteine residues in unfolded proteins, a process that is driven by the gain in protein conformational stability upon folding. Indeed, the folding of both ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) proceeded efficiently using GSeSeG as an oxidant, in the former case with a 2-fold rate increase relative to GSSG and in the latter case accelerating conversion of a stable folding intermediate to the native state. In addition, GSeSeG can also oxidize the common biological cofactor NADPH and is a good substrate for the NADPH-dependent enzyme glutathione reductase (kcat = 69 +/- 2 s-1, Km = 54 +/- 7 microM), suggesting that diselenides can efficiently interact with the cellular redox machinery. Surprisingly, the greater thermodynamic stability of diselenide bonds relative to disulfide bonds is not matched by a corresponding decrease in reactivity.

Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

Fatty acid synthases are dynamic ensembles of enzymes that can efficiently biosynthesize long hydrocarbon chains. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and β-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and MD simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli. The E. coli fatty acid synthase (FAS) produces fatty acids through an iterative cycle via the

Site creation: Enantioselective artificial metalloenzymes have been created by grafting a new active site onto bovine pancreatic polypeptide through the introduction of an amino acid capable of coordinating a copper(II) ion. This hybrid catalyst gave good enantioselectivities in the Diels-Alder and Michael addition reactions in water (see scheme) and displayed a very high substrate selectivity.

In the fatty acid biosynthesis of plants and bacteria, the acyl carrier protein (ACP) is known to sequester elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper we exploit solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. Addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity is confirmed by demonstration of a dual solvatochromic-crosslinking probe and solution-phase NMR. The chain-flipping mechanism can be visualized by single molecule fluorescent techniques, demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.