Structural and functional characterization of the triplet acyl carrier protein in the curacin cluster and its interaction partners

According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin. PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases. To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites. The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent. In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface. In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process

PHENOTYPIC RESPONSES of green alga CHLAMYDOMONAS REINHARDTII to nanoscale zero valent iron

S.Y. YEAP C, H.A. NGUYEN N, Blifernez-Klassen O, et al. PHENOTYPIC RESPONSES of green alga CHLAMYDOMONAS REINHARDTII to nanoscale zero valent iron. In: NANOCON 2022 Conference Proeedings. NANOCON Conference Proeedings. Vol 2022. TANGER Ltd.; 2022: 236-241.The toxicity of two iron-based nanoparticles: nFe3O4, and nZVI were assessed on freshwater microalgae, Chlamydomonas reinhardtii CC-5325. Microalgae response (total chlorophyll/carotenoids content, photosystem II efficiency, cell shape and total viable cell numbers) to the nanoparticles exposure (100 mg/L) was monitored up to 120 hours. Based on phytochrome and photosystem II analysis performed, almost no significant impact was found. However, microscopic analysis and the total viable cell numbers revealed a certain degree of inhibition effect showing altered cell shape, and higher number of dead cells after the exposure to both nanomaterials. The dead cell numbers increased within one hour after the exposure to nFe3O4, while nZVI caused rather slow inhibition effect and persisted until 48 h with the highest dead cell number. In a series of experiments performed, the results may justify that exposure of these two NPs initially slightly inhibited C. reinhardtii, however the culture was able to recover towards the end of the study, because of new cell generation and nZVI oxidation

PTS reaction to generate ST-gpD-Trx-His₆.

<p>A) Schematic representation of the <i>Npu</i> DnaE intein PTS reaction following the integration into the example protein ST-gpD-Trx-His₆. B) Amino acid sequences at the splice junction in the linker region of ST-gpD-Trx-His₆. Linker amino acids that differ from the original sequence (WT AA-sequence) are shown in blue. C) Western blot analysis of the <i>in vivo</i> PTS reaction to assemble ST-gpD-Trx-His₆. The theoretical molecular masses of the proteins are as follows: Splice product (<b>SP</b>) = 26.5–26.9 kDa; N-terminal precursor protein (<b>N’part</b>) = 25.0–25.2 kDa; C-terminal precursor protein (<b>C′part</b>) = 17.4–17.6 kDa; N-terminal hydrolysis product – ST-gpD (<b>N′hydro</b>) = 13.2–13.4 kDa. (pos = full length ST-gpD-Trx-His₆)_.</p

Principle of PTS and SPLICEFINDER.

<p>(A) The PTS reaction scheme. B) Schematic representation of the PTS cassette amplification and insertion procedure. The point of integration into the target gene (gene of interest; <i>GOI</i>) is controlled by the sequence of the PCR primers used for PTS cassette amplification. The integration of the PTS cassettes can be achieved in two ways: approach <b>1)</b> uses homologous recombination in <i>S. cerevisiae</i> and approach <b>2)</b> is based upon restriction-free (RF) cloning. Notably, in both approaches the amino acids flanking the intein can be adapted by the primer sequence. C) The PTS cassettes constructed and used in this study. (SP = splice product).</p

Integration of the <i>Ssp</i> DnaB intein cassette into the NRPS module Gramicidin S Synthetase B1.

<p>A) Schematic representation of the PTS reaction following the integration of the <i>Ssp</i> DnaB intein into GrsB1. B) Amino acid sequences at the splice junction at position 961. Deviations after splicing from the original sequence (WT AA-sequence) are shown in blue. C) (left) − SDS-PAGE analysis of the individually and dual induced combination GrsB1 ssp2; (right) SDS-PAGE analysis of purified splice products (<b>SP</b>) (combinations GrsB1 ssp2–4). The theoretical molecular masses of the proteins are as follows: N-terminal precursor protein (<b>N’part</b>)<b> = </b>124.9 kDa; Splice product (<b>SP</b>) = 124.2 kDa; C-terminal precursor protein (<b>C’part</b>) = 16.7 kDa. Impurities (<b>*</b>) are indicated. D) Activity assay for the GrsB1 splice products assembled through <i>in vivo</i> PTS with the <i>Ssp</i> DnaB intein. Analytical HPLC chromatograms (absorbance at 210 nm) are shown. The trace for the positive control, the full length wild type (WT) GrsB1 is shown in black. The peak at around 18.5 min was assigned via MS to the cyclic dipeptide D-Phe-L-Pro-diketopiperazine (DKP). When one of the substrates is omitted (ATP - yellow trace; Pro - grey trace; Phe - red trace) no product formation is detected. All purified GrsB1 splice products show DKP formation (GrsB1 ssp2–4).</p

Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP_I-ACP_II-ACP_III) within the CurA PKS module. We have determined the structure of the isolated holo-ACP_I and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP_I, which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP_I and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP_I-ACP_II-ACP_III tridomain, our data provide insight into the specificity of the chlorination reaction

Splicefinder : a fast and easy screening method for active protein trans-splicing positions

Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins