Characterization of recombinant Arabidopsis thaliana threonine synthase

Threonine synthase (TS) catalyses the last step in the biosynthesis of thre onine, the pyridoxol 5'-phosphate dependent conversion of L-homoserine phos phate (HSerP) into L-threonine and inorganic phosphate. Recombinant Arabido psis 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 acti vity was increased up to 85-fold by S-adenosyl-L-methionine (SAM) and speci fically 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, w hile the E. coli and yeast enzymes are monomers. The N-terminal region of a TS is essential for its regulatory properties and protects against inhibiti on by HSerP analogues, as an aTS devoid of 77 N-terminal residues was neith er activated by SAM nor inhibited by AMP, but was inhibited by HSerP analog ues. 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 concl usions are supported by a multiple amino-acid sequence alignment, which rev ealed the existence of two TS subfamilies. aTS was classified as a member o f subfamily 1 and its N-terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E, coli and yeast TS an members of subfamily 2, characterized by C-termini extending about 50 residues over those of sub family 1 members. As a first step towards a better understanding of the pro perties of aTS, the enzyme was crystallized by the sitting drop vapour diff usion method. The crystals diffracted to beyond 0.28 nm resolution and belo nged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, alpha = beta = gamma = 90 degrees).