Sulfonyl 3-Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase (original) (raw)
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Protein Science, 2007
Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 6 0.09 Å for the backbone atoms (1.21 6 0.13 Å for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 6 0.10 Å (0.99 6 0.11 Å for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.
Angewandte Chemie International Edition, 2016
In fungal non-reducing polyketide synthases (NR-PKS), the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanismbased crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-offunction ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides. Keywords Non-reducing polyketide synthase; crosslinking; acyl-carrier protein; product template domain Fungal polyketide natural products are chemically complex small molecules, many of which have diverse biological activities. Examples include the hepatocellular carcinogen aflatoxin
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.
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.
Gating mechanism of elongating β-ketoacyl-ACP synthases
Nature Communications, 2020
Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursi...
We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanismbased inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme-inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl-substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.
Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase
Bioorganic & Medicinal Chemistry Letters, 2008
The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.
Structure, 2000
Background: Holo-(acyl carrier protein) synthase (AcpS), a member of the phosphopantetheinyl transferase superfamily, plays a crucial role in the functional activation of acyl carrier protein (ACP) in the fatty acid biosynthesis pathway. AcpS catalyzes the attachment of the 4′-phosphopantetheinyl moiety of coenzyme A (CoA) to the sidechain of a conserved serine residue on apo-ACP. Results: We describe here the first crystal structure of a type II ACP from Bacillus subtilis in complex with its activator AcpS at 2.3 Å. We also have determined the structures of AcpS alone (at 1.8 Å) and AcpS in complex with CoA (at 1.5 Å). These structures reveal that AcpS exists as a trimer. A catalytic center is located at each of the solvent-exposed interfaces between AcpS molecules. Site-directed mutagenesis studies confirm the importance of trimer formation in AcpS activity. Conclusions: The active site in AcpS is only formed when two AcpS molecules dimerize. The addition of a third molecule allows for the formation of two additional active sites and also permits a large hydrophobic surface from each molecule of AcpS to be buried in the trimer. The mutations Ile5→Arg, Gln113→Glu and Gln113→Arg show that AcpS is inactive when unable to form a trimer. The co-crystal structures of AcpS-CoA and AcpS-ACP allow us to propose a catalytic mechanism for this class of 4′-phosphopantetheinyl transferases.