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.

Structure and Function of Cyanobacterial DHDPS and DHDPR

“…Likewise, inspection of the allosteric site confirms the presence of Glu at position 58 (His56 in Ec-DHDPS), but also reveals that Trp occupies position 55 (His53 in Ec-DHDPS), which is common in plants but not bacteria (Fig. 8C)26272829. The presence of a Trp at this positon is likely to explain the plant-like lysine IC 50 for Av-DHDPS (Fig.…”

“…The structures of DHDPS from several plant species have also been determined showing that the plant orthologues also form homotetramers but in a ‘back-to-back’ dimer-of-dimers arrangement (Fig. 1B)26272829. Additionally, there are also reports of dimeric DHDPS enzymes from P. aeruginosa 21, S. aureus 2223, and Shewanella benthica 30.…”

“…However, the structure of a plant DHDPR has not yet been determined, but a recent study employing small angle X-ray scattering suggests that the enzyme from Arabidopsis thaliana adopts a novel dimeric structure (Fig. 1C)28. Accordingly, there appears to be structural diversity between bacterial and plant orthologues of both DHDPS and DHDPR.…”

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

“…This rate-limiting step is catalysed by the enzyme DHDPS. In all Gram-negative pathogens (Dobson, Devenish et al, 2005;Phenix & Palmer, 2008;Devenish et al, 2009;Kaur et al, 2011) and plants (Matthews & Widholm, 1979;Wallsgrove & Mazelis, 1980;Kumpaisal et al, 1987;Dereppe et al, 1992;Blickling, Beisel et al, 1997;Griffin et al, 2012;Atkinson et al, 2013) studied to date, lysine acts as a feedback inhibitor to DHDPS.…”

“…This gives rise to four active sites in a tetrameric DHDPS or two active sites in a dimeric DHDPS (Mirwaldt et al, 1995). Adjacent to the active sites is a cleft at the tight-dimer interface to which lysine binds in order to inhibit the enzyme (Dobson, Griffin et al, 2005;Kaur et al, 2011;Griffin et al, 2012;Atkinson et al, 2013).…”