A new enzyme superfamily ? the phosphopantetheinyl transferases (original) (raw)
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Catalytic self-acylation of type II polyketide synthase acyl carrier proteins
Chemistry & Biology, 1998
Background: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes-the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in viva. Results: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin Cl 7s holo-ACP has a K, for malonyl CoA.of 219 PM and a kcat of 0.34 min-'. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate .of malonylation.
Chemistry & Biology, 2006
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.
Structure, 2001
catalyzed by -ketoacyl synthase (KAS) enzymes. This reaction takes place in three steps (Figure 1), as follows: transfer of an acyl carrier protein (ACP) bound primer to the active site cysteine; decarboxylation of the donor substrate, malonyl-ACP, to give an ACP bound carbanion; and condensation of this carbanion with the enzyme bound primer substrate. While early studies educed re-Denmark † Department of Genetics sults supporting the hypothesis that the Claisen condensation is a concerted reaction [1], subsequent work sug-Institute of Molecular Biology University of Copenhagen gests a stepwise reaction is more likely [2-4]. Repetitive condensations yield long carbon chains like those char-Copenhagen, DK-1353 Denmark acterizing fatty acids, polyketide antibiotics, flavonoids, toxins, and cell wall components such as waxes and mycolic acids. Most frequently, these condensing enzymes are components of multienzyme complexes that, Summary in addition to the condensation reaction, activate the primer and donor substrates as well as prepare the Background: -ketoacyl-acyl carrier protein synthase growing carbon chain for the next elongation cycle. (KAS) I is vital for the construction of the unsaturated Varying the nature of the substrates, how they are actifatty acid carbon skeletons characterizing E. coli memvated, and which additional reactions take place bebrane lipids. The new carbon-carbon bonds are created tween each condensation (Figure 1) results in the broad by KAS I in a Claisen condensation performed in a threerange of end products noted above from different biostep enzymatic reaction. KAS I belongs to the thiolase chemical pathways. Extensive variation within each fold enzymes, of which structures are known for five type of end product (for example, fatty acids) is also other enzymes. possible. How this is achieved depends in part on the nature of the participating enzyme complex. Two basic Results: Structures of the catalytic Cys-Ser KAS I mutypes are known, those composed of one or two polytant with covalently bound C10 and C12 acyl substrates functional proteins coded for by the corresponding numhave been determined to 2.40 and 1.85 Å resolution, ber of genes (type I) and those consisting of monofuncrespectively. The KAS I dimer is not changed by the tional proteins that are each encoded by discrete genes formation of the complexes but reveals an asymmetric (type II). Different end products can be realized with binding of the two substrates bound to the dimer. A type I complexes if additional monofunctional proteins detailed model is proposed for the catalysis of KAS I. are present; for example, a second thioesterase is capa-Of the two histidines required for decarboxylation, one ble of taking C8-C12 fatty acyl chains before they are donates a hydrogen bond to the malonyl thioester oxo completely elongated to C14-C18 from the rat fatty acid group, and the other abstracts a proton from the leaving synthase (FAS) complex [5]. Isozymes of components group.
New Start and Finish for Complex Polyketide Biosynthesis
Chemistry & Biology, 2004
as well as a loading module for transferring the starter acyl group onto the first KS domain. This modular orga-Biosynthesis nization allows programmed assembly of a defined sequence of starter and extender units, together with controlled processing of each -ketone group. The final The polyketide vicenistatin has significant anticancer product may be cyclized by a thioesterase (TE) to give activity. In the January issue of Chemistry & Biology, a macrolactone.
A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis
Nature Chemical Biology, 2013
Type I PKSs often utilise programmed β-branching, via enzymes of an "HMG-CoA synthase (HCS) cassette", to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules. Polyketide synthases (PKSs) 1 are responsible for an extraordinarily large and diverse group of natural products that have important pharmaceutical applications such as antibiotic, antitumor, antifungal, anticholesterolemic and antiparasitic agents 2. PKSs are classified on the basis of their protein architecture; bacterial type I PKSs are large multifunctional polypeptides with all core enzymatic functions for elongation and modification of the carbon backbone grouped as modules. Type I PKS biosynthetic pathways are normally constructed with one module for each condensation reaction, often with additional modules that can make non-elongating modifications, or iterative modifications incorporating multiple units. The minimal functions in an elongating module are the ketosynthase (KS) domain, which acquires either a starter unit or the oligoketide from the previous module, and an acyl carrier protein (ACP) domain that holds the extender unit (most commonly malonate or methylmalonate). The KS catalyses a Claisen condensation, creating a new carbon-carbon bond in the ACP bound intermediate. Canonically, type I modules also contain an acyltransferase (AT) that loads the extender unit onto the ACP (known as cis-AT systems). However, an increasing number of known Type I PKSs, including pathways considered here, are trans-AT systems which lack integral AT domains in the extension modules but that encode one or more separate ATs to perform this function 3. After the Claisen condensation, modifications of the acyl chain may take place before transfer to the next module, most commonly β-ketoreduction (KR) alone, KR and dehydration (DH) or KR, DH and enoyl reduction (ER), yielding hydroxyl, alkene and alkane moieties respectively. In addition, trans-AT systems can introduce α-methyl groups with a methyl-transferase (MT) domain as part of the Type I module. A more complex modification found in numerous known trans-AT and several cis-type I systems occurs at the βketo group and is introduced by a transacting "HCS cassette", typically comprising an ACP, an hydroxymethylglutaryl-CoA (HMG-CoA) synthase (HCS), two proteins belonging to the crotonase superfamily and a decarboxylase. The HCS cassettes can introduce otherwise difficult modifications such as β-methyl, β-ethyl, β-methoxymethyl, cyclopropane and vinyl chloride moieties in different systems 4. This cassette normally acts on a type I module characterised by the absence of KR, DH or ER functions, often with tandemly repeated ACPs. In the myxovirescin system there are two HCS enzymes, one of which acts specifically at one of two β-branch ACPs. Understanding what signals such specific modifications is
Biochemistry, 2000
Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO 2. The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2′-(5′′-triphosphoribosyl)-3′-dephospho-CoA. In this reaction, a new R (1′′ f 2′) glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5′-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2′-(5′′-triphosphoribosyl)-3′-dephospho-CoA prosthetic group precursor. This complex, called MdcG i , is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG i with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2′-(5′′-triphosphoribosyl)-3′dephospho-CoA:apo ACP 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2′-(5′′-triphosphoribosyl)-3′-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.
Journal of the American Chemical Society, 2013
Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein−protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein−protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.