Antibacterial activity of cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr) from <i>Streptomyces</i> sp<i>.</i> strain 22-4 against phytopathogenic bacteria

<p>Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of <i>Streptomyces</i> sp. strain 22-4 and tested against three economically important plant pathogens, <i>Xanthomonas axonopodis</i> pv. citri, <i>Ralstonia solanacearum</i> and <i>Clavibacter michiganensis</i>. Both cyclic dipeptides were active against <i>X. axonopodis</i> pv. citri and <i>R. Solanacearum</i> with MIC of 31.25 <i>μ</i>g/mL<i>.</i> No activity could be observed against <i>C. michiganensis</i>.</p

SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein–thioesterase domains from a fungal non‐reducing polyketide synthase

Menisporopsin A is a fungal bioactive macrocyclic polylactone requiring only reducing (R) and non-reducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions with 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-aString 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 optimised polyketide scaffolds

Solution Structures of the Acyl Carrier Protein Domain from the Highly Reducing Type I Iterative Polyketide Synthase CalE8

Biosynthesis of the enediyne natural product calicheamicins γ1I in Micromonospora echinospora ssp. calichensis is initiated by the iterative polyketide synthase (PKS) CalE8. Recent studies showed that CalE8 produces highly conjugated polyenes as potential biosynthetic intermediates and thus belongs to a family of highly-reducing (HR) type I iterative PKSs. We have determined the NMR structure of the ACP domain (meACP) of CalE8, which represents the first structure of a HR type I iterative PKS ACP domain. Featured by a distinct hydrophobic patch and a glutamate-residue rich acidic patch, meACP adopts a twisted three-helix bundle structure rather than the canonical four-helix bundle structure. The so-called ‘recognition helix’ (α2) of meACP is less negatively charged than the typical type II ACPs. Although loop-2 exhibits greater conformational mobility than other regions of the protein with a missing short helix that can be observed in most ACPs, two bulky non-polar residues (Met992, Phe996) from loop-2 packed against the hydrophobic protein core seem to restrict large movement of the loop and impede the opening of the hydrophobic pocket for sequestering the acyl chains. NMR studies of the hydroxybutyryl- and octanoyl-meACP confirm that meACP is unable to sequester the hydrophobic chains in a well-defined central cavity. Instead, meACP seems to interact with the octanoyl tail through a distinct hydrophobic patch without involving large conformational change of loop-2. NMR titration study of the interaction between meACP and the cognate thioesterase partner CalE7 further suggests that their interaction is likely through the binding of CalE7 to the meACP-tethered polyene moiety rather than direct specific protein-protein interaction

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

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

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

\ua9 2020 Federation of European Biochemical Societies. 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

Recognition of intermediate functionality by acyl carrier protein over a complete cycle of fatty acid biosynthesis

SummaryIt remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis

Proposed arrangement of proteins forming a bacterial type II polyketide synthase.

SummaryAklanonic acid is synthesized by a type II polyketide synthase (PKS) composed of eight protein subunits. The network of protein interactions within this complex was investigated using a yeast two-hybrid system, by coaffinity chromatography and by two different computer-aided protein docking simulations. Results suggest that the ketosynthase (KS) α and β subunits interact with each other, and that the KSα subunit also probably interacts with a malonyl-CoA:ACP acyltransferase (DpsD), forming a putative minimal synthase. We speculate that DpsD may physically inhibit the priming reaction, allowing the choice of propionate rather than acetate as the starter unit. We also suggest a structural role for the cyclase (DpsY) in maintaining the overall structural integrity of the complex

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 ACP 1-ACP 2), 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 ACP 1-ACP 2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP 1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP 1-ACP 2 by NMR revealed conformational exchange found predominantly in the ACP 2 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 ACP 1-ACP 2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP 1 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. </p