Characterization of recombinant Arabidopsis thaliana threonine synthase

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Abstract
Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5′‐phosphate dependent conversion of l‐homoserine phosphate (HSerP) into l‐threonine and inorganic phosphate. Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast. This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85‐fold by S‐adenosyl‐l‐methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E. coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E. coli and yeast enzymes are monomers. The N‐terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N‐terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues. The C‐terminal region of aTS seems to be involved in dimer formation, as the N‐terminally truncated aTS was also found to be a dimer. These conclusions are supported by a multiple amino‐acid sequence alignment, which revealed the existence of two TS subfamilies. aTS was classified as a member of subfamily 1 and its N‐terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E. coli and yeast TS are members of subfamily 2, characterized by C‐termini extending about 50 residues over those of subfamily 1 members. As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method. The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, α = β = γ = 90°).