Structure–function analyses of two plant meso-diaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control

Crowther, Jennifer M.; Cross, Penelope J.; Oliver, Melody; Leeman, Mary M.; Bartl, Austin J.; Weatherhead, Anthony W.; North, Rachel A.; Donovan, Katherine A.; Griffin, Michael D. W.; Suzuki, Hisao; Hudson, André O.; Kasanmascheff, Müge; Dobson, Renwick C. J.

doi:10.1074/jbc.ra118.006825

Cited by 6 publications

(8 citation statements)

References 31 publications

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“…For example, only the D-primed seedlings increased compounds such as β-solanine, guaiacol or plant hormones-related compounds such as gibberellin 34, the brassinosteroids 6-deoxo-24-epicathasterone and campest-5-en-3-one, the sugar maltose or some flavones such as baicalin and 7-hydroxyflavone, among others ( Supplementary Table 6 ). However, C but not D increased certain sesquiterpenoids such as curcuquinone or the main precursor for the synthesis of the aromatic amino acids, shikimate, relevant pathway controlling plant growth and development ( Tzin and Galili, 2010 ), or the metabolite meso -diaminopimelate, substrate for the synthesis of L-lysine ( Crowther et al, 2019 ) ( Supplementary Table 5 ).…”

Section: Resultsmentioning

confidence: 99%

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses

et al. 2021

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The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 μl ml–1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.

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Section: Resultsmentioning

confidence: 99%

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses

et al. 2021

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“…The penultimate step of the DAP pathway involves the formation of meso ‐DAP, which is catalysed by DAP epimerase (DAPEpi, EC 5.1.1.7) [16]. Ultimately, meso ‐DAP is decarboxylated by DAP decarboxylase (DAPDC, EC 4.1.1.20) to form l ‐lysine (hereinafter referred to as lysine) [17,18].…”

Section: Introductionmentioning

confidence: 99%

Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms

Hall

Boarder

et al. 2021

The FEBS Journal

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Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate‐limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l‐lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing > 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine‐mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10‐fold more sensitive to lysine. We subsequently used X‐ray crystallography to probe for structural differences between the apo‐ and lysine‐bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6‐fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. Databases Structures described are available in the Protein Data Bank under the accession numbers https://doi.org/10.2210/pdb6VVH/pdb and https://doi.org/10.2210/pdb6VVI/pdb.

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“…The enzyme has a dimeric structure in which the C-terminus of one monomer is wrapped around the other ( Fig. S3 ), a topology that is conserved in related PLP-dependent fold-type III enzymes ( Crowther et al. , 2019 ).…”

Section: Resultsmentioning

confidence: 99%

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase

Marjanovic

Ramírez-Palacios

Masman

et al. 2021

Protein Engineering, Design and Selection

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Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes.

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Structure–function analyses of two plant meso-diaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control

Cited by 6 publications

References 31 publications

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses

Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase

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