Deciphering the Function and Structure of PA1216 as an S‐Adenosyl‐l‐Methionine Binding Protein Using Differential Scanning Fluorimetry and Circular Dichroism
Jul 2026· Protein Science· Vol 35· 0 citations· 47 references
Medicine
TL;DR
PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1 is characterized and a platform for characterizing cryptic gene clusters within secondary metabolic pathways is established for characterizing cryptic gene clusters within secondary metabolic pathways.
Abstract
Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration‐dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high‐throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl‐glycine dipeptides, amino esters with aromatic or basic side chains, and N‐Boc‐protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann‐like fold and a glycine‐rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.
RNA methyltransferases (MTases) play a significant role in post-transcriptional regulation in bacteria, significantly influencing virulence, stress adaptation and host-pathogen interactions. In Mycobacterium tuberculosis, several putative MTases remain functionally uncharacterized. This study aims to elucidate the structural, biophysical and functional properties of two predicted M. tuberculosis RNA MTases, Rv3366 and Rv3919c, to better understand their roles in mycobacterial physiology and pathogenicity. The genes encoding Rv3366 and Rv3919c were cloned, heterologously expressed in Escherichia coli and purified using affinity chromatography. Enzymatic activity was evaluated using a methyltransferase-Glo™ assay with S-adenosylmethionine (SAM) as the methyl donor. Biophysical and structural characterization was carried out using circular dichroism (CD) spectroscopy, fluorescence spectroscopy and thermal shift assays. Both proteins exhibited SAM-dependent MTase activity along with appreciable RNA-binding affinity. CD spectral analysis revealed a predominantly α-helical secondary structure, while fluorescence and thermal shift assays confirmed proper folding and notable thermal stability. Collectively, Rv3366 and Rv3919c display hallmark characteristics of functional RNA MTases, suggesting their involvement in RNA modification pathways that may contribute to M. tuberculosis virulence and adaptation. These findings establish a framework for future studies to explore their mechanistic roles and evaluate their potential as therapeutic targets.
Tasmin Nazim, Faraz Ahmed, M. Beg et al.· Journal of Biomolecular Stru...· 0 citations
Genome mining of the marine-derived Streptomyces sp. S42 uncovered a type II polyketide synthetase (T2 PKS) biosynthetic gene cluster (BGC) harboring a gene for 3-ketoacyl-ACP synthase III (KAS III), a hallmark of non-acetate starter unit incorporation, suggesting that the BGC may produce previously unidentified aromatic polyketides. Heterologous expression and promoter engineering of this prioritized BGC in host Streptomyces albus J1074 activated the biosynthetic pathway, leading to the isolation of eight new polycyclic aromatic derivatives, verrucones A–H (1–8). Comprehensive structural elucidation via NMR and HRESIMS revealed that these compounds feature either a 2-methylbutyryl or an isobutyryl starter unit and can be classified into three distinct skeletal types. Based on these findings and bioinformatic analysis, a plausible biosynthetic pathway for 1–8 involving divergent spontaneous cyclization from a common nascent polyketide intermediate was proposed. Among the isolated compounds, 1–5 exhibited inhibitory activity against several protein tyrosine phosphatases (PTPs) with IC50 values ranging from 1.84 μM to 24.82 μM. This study presents a successful case study demonstrating that combining KAS III-targeted genome mining with heterologous expression is a viable approach for discovering non-acetate-primed aromatic polyketides.
Xingkun Hao, Ming Yang, Ping Yan et al.· Microorganisms· 0 citations
It is indicated that combining A-domain substrate prediction with genome mining is an effective strategy for discovering NRPs in Aspergillus, expanding the understanding of their biosynthetic potential.
Huawei Lv, Zhi-Yan Zhang, Jia-Gui Tang et al.· Archives of Microbiology· 0 citations
This work identifies the putative myriocin biosynthesis gene cluster (BGC) through de novo sequencing of two producing fungi, Isaria sinclairii and Mycelia sterilia, yielding genomes of 27 and 20 secondary metabolite BGCs, respectively.
B. Rutter, Michael A. Herrera, Gustavo Perez Ortiz et al.· Communications Biology· 0 citations
Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all-α-helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.
Results identify the B37 vdcBCD cluster as a bifunctional aromatic acid decarboxylation module and link this reaction to the formation of phenolic intermediates relevant to yellow-stained cork and cork taint-associated chemistry, including the de novo formation of chlorophenols and chloroanisoles.
M. Ruiz-Muñoz, R. Cobos, Carla Calvo-Peña et al.· Frontiers in Microbiology· 0 citations
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