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Crystal structure of thermospermine synthase from Medicago truncatula and substrate discriminatory features of plant aminopropyltransferases.

ABSTRACT: Polyamines are linear polycationic compounds that play a crucial role in the growth and development of higher plants. One triamine (spermidine, SPD) and two tetraamine isomers (spermine, SPM, and thermospermine, TSPM) are obtained by the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine and SPD. These reactions are catalyzed by the specialized aminopropyltransferases. In that respect, plants are unique eukaryotes that have independently evolved two enzymes, thermospermine synthase (TSPS), encoded by the gene ACAULIS5, and spermine synthase, which produce TSPM and SPM, respectively. In this work, we structurally characterize the ACAULIS5 gene product, TSPS, from the model legume plant Medicago truncatula (Mt). Six crystal structures of MtTSPS - one without ligands and five in complexes with either reaction substrate (SPD), reaction product (TSPM), or one of three cofactor analogs (5'-methylthioadenosine, S-adenosylthiopropylamine, and adenosine) - give detailed insights into the biosynthesis of TSPM. Combined with small-angle X-ray scattering data, the crystal structures show that MtTSPS is a symmetric homotetramer with an interdomain eight-stranded β-barrel. Such an assembly and the presence of a hinge-like feature between N-terminal and C-terminal domains give the protein additional flexibility which potentially improves loading substrates and discarding products after the catalytic event. We also discuss the sequence and structural features around the active site of the plant aminopropyltransferases that distinguish them from each other and determine their characteristic substrate discrimination.

SUBMITTER: Sekula B

PROVIDER: S-EPMC7983153 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUnderstanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities.ResultsBacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment × soil origin and compartment × plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless, all compartments were dominated by Ensifer, the genus of rhizobia that forms root nodule symbiosis with M. truncatula, and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere.ConclusionsOur results demonstrate strong host filtering effects, with rhizospheres driven by soil origin and internal plant compartments driven by host genetics, and identify several key nodule-inhabiting taxa that coexist with rhizobia in the native range. Our results set the stage for future functional genetic experiments aimed at expanding our pairwise understanding of legume-rhizobium symbiosis toward a more mechanistic understanding of plant microbiomes. Video Abstract.

Project description:One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

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Crystal structure of thermospermine synthase from Medicago truncatula and substrate discriminatory features of plant aminopropyltransferases.

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