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Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

ABSTRACT: In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

SUBMITTER: Griffin MD

PROVIDER: S-EPMC3390394 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

Griffin Michael D W MD Billakanti Jagan M JM Wason Akshita A Keller Sabrina S Mertens Haydyn D T HD Atkinson Sarah C SC Dobson Renwick C J RC Perugini Matthew A MA Gerrard Juliet A JA Pearce Frederick Grant FG

PloS one 20120705 7

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. ...[more]

PMID: 22792278

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Project description:BackgroundIn all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant Arabidopsis thaliana, to understand the function of nucleoside modifications in plant growth and development.ResultsIn this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, Arabidopsis thaliana and hybrid aspen (Populus tremula × tremuloides). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in Sacccharomyces cerevisiae and Escherichia coli, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family.Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m1G, m2G, m7G and ncm5U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm5U modification). The AtTRM11 mutant exhibited an early-flowering phenotype, and the AtELP1 mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots.ConclusionsUsing a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in Arabidopsis thaliana. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.

Project description:Biosynthesis of chlorophyll (Chl) involves many enzymatic reactions that share several first steps for biosynthesis of other tetrapyrroles such as heme, siroheme, and phycobilins. Chl allows photosynthetic organisms to capture light energy for photosynthesis but with simultaneous threat of photooxidative damage to cells. To prevent photodamage by Chl and its highly photoreactive intermediates, photosynthetic organisms have developed multiple levels of regulatory mechanisms to coordinate tetrapyrrole biosynthesis (TPB) with the formation of photosynthetic and photoprotective systems and to fine-tune the metabolic flow with the varying needs of Chl and other tetrapyrroles under various developmental and environmental conditions. Among a wide range of regulatory mechanisms of TPB, this review summarizes transcriptional regulation of TPB genes during plant development, with focusing on several transcription factors characterized in Arabidopsis thaliana. Key TPB genes are tightly coexpressed with other photosynthesis-associated nuclear genes and are induced by light, oscillate in a diurnal and circadian manner, are coordinated with developmental and nutritional status, and are strongly downregulated in response to arrested chloroplast biogenesis. LONG HYPOCOTYL 5 and PHYTOCHROME-INTERACTING FACTORs, which are positive and negative transcription factors with a wide range of light signaling, respectively, target many TPB genes for light and circadian regulation. GOLDEN2-LIKE transcription factors directly regulate key TPB genes to fine-tune the formation of the photosynthetic apparatus with chloroplast functionality. Some transcription factors such as FAR-RED ELONGATED HYPOCOTYL3, REVEILLE1, and scarecrow-like transcription factors may directly regulate some specific TPB genes, whereas other factors such as GATA transcription factors are likely to regulate TPB genes in an indirect manner. Comprehensive transcriptional analyses of TPB genes and detailed characterization of key transcriptional regulators help us obtain a whole picture of transcriptional control of TPB in response to environmental and endogenous cues.

Dataset Information

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

Publications

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

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