Direct structural analysis of a single acyl carrier protein domain in fatty acid synthase from the fungus Saccharomyces cerevisiae.

Acyl carrier protein (ACP) is the work horse of polyketide (PKS) and fatty acid synthases (FAS) and acts as a substrate shuttling domain in these mega enzymes. In fungi, FAS forms a 2.6 MDa symmetric assembly with six identical copies of FAS1 and FAS2 polypeptides. However, ACP spatial distribution is not restricted by symmetry owing to the long and flexible loops that tether the shuttling domain to its corresponding FAS2 polypeptide. This symmetry breaking has hampered experimental investigation of substrate shuttling route in fungal FAS. Here, we develop a protein engineering and expression method to isolate asymmetric fungal FAS proteins containing odd numbers of ACP domains. Electron cryomicroscopy (cryoEM) observation of the engineered complex reveals a non-uniform distribution of the substrate shuttling domain relative to its corresponding FAS2 polypeptide at 2.9 Å resolution. This work lays the methodological foundation for experimental study of ACP shuttling route in fungi.

Amino acid (acyl carrier protein) ligase-associated biosynthetic gene clusters reveal unexplored biosynthetic potential.

This study aimed to evaluate the postulated cellular function of a novel family of amino acid (acyl carrier protein) ligases (AALs) in natural product biosynthesis. Here, we analyzed the manually curated, putative, aal-associated natural product biosynthetic gene clusters (NP BGCs) using two computational platforms for NP prediction, antiSMASH-BiG-SCAPE-CORASON and DeepBGC. The detected BGCs included a diversity of type I polyketide/nonribosomal peptide (PKS/NRPS) hybrid BGCs, exemplified by the guadinomine BGC, which suggested a dedicated function of AALs in the biosynthesis of rare (2S)-aminomalonyl-ACP extension units. Besides modular PKS/NRPSs and NRPSs, AAL-associated BGCs were predicted to assemble arylpolyenes, ladderane lipids, phosphonates, aminoglycosides, ß-lactones, and thioamides of both nonribosomal and ribosomal origins. Additionally, we revealed a frequent association of AALs with putative, seldom observed transglutaminase-like and BtrH-like transferases of the cysteine protease superfamily, which may form larger families of ACP-dependent amide bond catalysts used in NP synthesis. Our results disclosed an exceptional chemical novelty and biosynthetic potential of the AAL-associated BGCs in NP biosynthesis. The presented in silico evidence supports the initial hypothesis and provides an important foundation for future experimental studies aimed at discovering novel pharmaceutically relevant active compounds.

Tracing of Acyl Carrier Protein-channeled Mitomycin Intermediates in Streptomyces caespitosus Facilitates Characterization of the Biosynthetic Steps for AHBA-GlcN Formation and Processing.

Mitomycins are a family of naturally occurring, potent alkylating agents in which the C member has been clinically used for cancer chemotherapy for over 5 decades. In Streptomyces caespitosus, mitomycins are derived from an N-glycoside composed of a 3-amino-5-hydroxybenzoic acid (AHBA) unit and a d-glucosamine (GlcN) unit; however, how this N-glycoside is formed and rearranged to a mitosane, for example, the compact polycyclic ring system of mitomycin C, remains elusive. Benefiting from the development of a method used to trace the mitomycin intermediates that accumulate on an acyl carrier protein (ACP), we here dissect the enzymatic steps for AHBA-GlcN formation and processing to underlie the mitosane structure. Following the N-glycosylation of AHBA with activated N-acetyl-GlcN, deacetylation occurs on ACP to provide AHBA-GlcN. Then, the sugar portion of this N-glycoside is transformed into a linear aminodiol that terminates with an epoxyethane, yielding an ACP-channeled intermediate that is ready for mitosane formation through crosslinking between the AHBA and linearized sugar units. This transformation is unusual and relies on the functional association of a dihydronicotinamide adenine dinucleotide (phosphate)-dependent protein with a radical S-adenosyl-l-methionine protein. Characterization of these ACP-based enzymatic steps for AHBA-GlcN formation and processing sheds light on the poorly understood biosynthetic pathway of mitomycins.

Protein-Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin.

The ketosynthase (KS) domain is a core domain found in modular polyketide synthases (PKSs). To maintain the polyketide biosynthetic fidelity, the KS domain must only accept an acyl group from the acyl carrier protein (ACP) domain of the immediate upstream module even when they are separated into different polypeptides. Although it was reported that both the docking domain-based interactions and KS-ACP compatibility are important for the interpolypeptide transacylation reaction in 6-deoxyerythronolide B synthase, it is not clear whether these findings are broadly applied to other modular PKSs. Herein, we describe the importance of protein-protein recognition in the intermodular transacylation between VinP1 module 3 and VinP2 module 4 in vicenistatin biosynthesis. We compared the transacylation activity and crosslinking efficiency of VinP2 KS4 against the cognate VinP1 ACP3 with the noncognate one. As a result, it appeared that VinP2 KS4 distinguishes the cognate ACP3 from other ACPs.

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin.

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein-protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein-protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein-protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein-thioesterase domains from a fungal nonreducing polyketide synthase.

Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP2 and ACP1 -ACP2 )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis. Small-angle X-ray scattering (SAXS) studies in combination with homology modelling reveal that these polypeptides adopt a distinctive beads-on-a-string configuration, characterized by the presence of highly flexible interdomain linkers. These models provide a platform for studying domain organization and interdomain interactions in fungal NR-PKSs, which may be of value in directing the design of functionally optimized polyketide scaffolds.

Structural modification of a novel inhibitor for mycobacterium enoyl-acyl carrier protein reductase assisted by In silico structure-based drug screening.

Background: Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (mtInhA) is involved in the biosynthesis of mycolic acids, a major component of mycobacterial cell walls, and has been targeted in the development of anti-tuberculosis (TB) drugs. In our previous in silico structure-based drug screening study, we identified KES4, a novel class of mtInhA inhibitor. KES4 is composed of four ring structures (A-D-rings) and molecular dynamic simulation predicted that the D-ring is essential for the interaction with mtInhA. Methods: The structure-activity relationship study of the D-ring was attempted and aided by in silico docking simulations to improve the mtInhA inhibitory activity of KES4. A virtual chemical library of the D-ring-modified KES4 was then constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. The candidate compound showing the highest GOLD score, referred to as KEN1, was synthesized, and its biological properties were compared with those of the lead compound KES4. Results: We achieved the synthesis of KEN1 and evaluated its effects on InhA activity, mycobacterial growth, and cytotoxicity. The antimycobacterial activity of KEN1 was comparable to that of the lead compound (KES4), although it exhibited superior activity in mtInhA inhibition. \. Conclusions: We obtained a KES4 derivative with high mtInhA inhibitory activity by in silico docking simulation with a chemical library consisting of a series of D-ring-modified KES4.

Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins.

The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the molecular interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron-sulfur (Fe-S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe-S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.

Colorimetric Assay Reports on Acyl Carrier Protein Interactions.

The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4'-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB-) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2-, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial agents by enabling the rapid identification of small molecule inhibitors of ACP-KS interactions.

Structure, function and dynamics in acyl carrier proteins.

Carrier proteins are four-helix bundles that covalently hold metabolites and secondary metabolites, such as fatty acids, polyketides and non-ribosomal peptides. These proteins mediate the production of many pharmaceutically important compounds including antibiotics and anticancer agents. Acyl carrier proteins (ACPs) can be found as part of a multi-domain polypeptide (Type I ACPs), or as part of a multiprotein complex (Type II). Here, the main focus is on ACP2 and ACP3, domains from the type I trans-AT polyketide synthase MmpA, which is a core component of the biosynthetic pathway of the antibiotic mupirocin. During molecular dynamics simulations of their apo, holo and acyl forms ACP2 and ACP3 both form a substrate-binding surface-groove. The substrates bound to this surface-groove have polar groups on their acyl chain exposed and forming hydrogen bonds with the solvent. Bulky hydrophobic residues in the GXDS motif common to all ACPs, and similar residues on helix III, appear to prohibit the formation of a deep tunnel in type I ACPs and type II ACPs from polyketide synthases. In contrast, the equivalent positions in ACPs from type II fatty acid synthases, which do form a deep solvent-excluded substrate-binding tunnel, have the small residue alanine. During simulation, ACP3 with mutations I61A L36A W44L forms a deep tunnel that can fully bury a saturated substrate in the core of the ACP, in contrast to the surface groove of the wild type ACP3. Similarly, in the ACP from E. coli fatty acid synthase, a type II ACP, mutations can change ligand binding from being inside a deep tunnel to being in a surface groove, thus demonstrating how changing a few residues can modify the possibilities for ligand binding.

In vitro characterization of MitE and MitB: Formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins.

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis.

Characterization of Acyl Carrier Protein-Dependent Glycosyltransferase in Mitomycin C Biosynthesis.

Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two building blocks, AHBA and GlcNAc, are coupled during biosynthesis remained uncharacterized. Here we report evidence that AHBA is first loaded onto an MmcB acyl carrier protein (ACP) by a MitE acyl ACP synthetase, followed by a transfer of GlcNAc from UDP-GlcNAc by MitB. The results suggest that the early steps of mitomycin biosynthesis proceed via intermediates linked to MmcB.

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold.

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

A conformational switch from a closed apo- to an open holo-form equips the acyl carrier protein for acyl chain accommodation.

Acyl carrier proteins (ACPs) play crucial roles in the biosynthesis of fatty acids, non-ribosomal polypeptides and polyketides. The three-dimensional NMR structure of Leishmania major holo-LmACP, belonging to the type II pathway, has been reported previously, but the structure of its apo-form and its conformational differences with the holo-form remain to be explored. Here we report the crystal structures of apo-LmACP (wild-type and S37A mutant) at 2.0 Šresolution and compare their key features with the structures of holo-LmACP (wild-type) and other type II ACPs from Escherichia coli and Plasmodium falciparum. The crystal structure of apo-LmACP, which is homologous to other type II ACPs, displays some key structural rearrangements as compared to its holo-structure. Contrary to holo-form, which exists predominantly as a monomer, the apo-form exists as a mixture of monomeric and dimeric population in solution. In contrast to the closed structure of apo-LmACP, holo-LmACP structure was observed in an open conformation as a result of reorganization of specific helices and loops. We propose that the structural changes exhibited by LmACP occur due to the attachment of the phosphopantetheine arm and may be a prerequisite for the initiation of fatty acid synthesis. The movement of helix 3 may also play a role in the dissociation of holo-LmACP from its cognate enzymes of the FAS II pathway.

Insights into ß-ketoacyl-chain recognition for ß-ketoacyl-ACP utilizing AHL synthases.

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

Architectural Features of Human Mitochondrial Cysteine Desulfurase Complexes from Crosslinking Mass Spectrometry and Small-Angle X-Ray Scattering.

Cysteine desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial cysteine desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the cysteine desulfurase-ISCU and cysteine desulfurase-ISCU-frataxin complexes. We present a structural model for the cysteine desulfurase-ISCU-frataxin complex derived from chemical crosslinking restraints in conjunction with the recent crystal structure of the cysteine desulfurase-ISCU-zinc complex and distance constraints from nuclear magnetic resonance.

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Visualizing the Adenylation Activities and Protein-Protein Interactions of Aryl Acid Adenylating Enzymes.

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.

Iron-sulfur cluster biogenesis and trafficking in mitochondria.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.