Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5 H -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, a = b = g = 908).Keywords: threonine synthase; pyridoxal 5 H -phosphate; S-adenosyl-l-methionine; enzyme activation; crystallization.Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5 H -phosphate dependent conversion of l-homoserine phosphate (HSerP; O-phospho-lhomoserine) into l-threonine and inorganic phosphate. TS has previously been purified and characterized from bacterial and fungal sources [1±5]. Much less, however, is known about the enzymology of higher plant TS. The enzyme is localized in chloroplasts [6] and is markedly stimulated by S-adenosylmethionine (SAM) [7±12]. SAM stimulation has been related to the regulation of higher plant threonine and methionine biosynthesis. In case of methionine overproduction-regulation is assumed to be exerted through its activated form, SAM, by synergistic feedback inhibition in the presence of lysine of aspartate kinase [13] and by activation of TS, thereby channelling HSerP from methionine into threonine biosynthesis. However, the significance of activation of TS by SAM as a regulatory mechanism for methionine biosynthesis in vivo is a matter of dispute [14±16].Escherichia coli TS (eTS) is the target enzyme for Z-2-amino-5-phosphono...

The second acylation reaction in glycerolipid biosynthesis is catalyzed by an sn-1 -acylglycerol-3-phosphate acyltransferase. The enzyme of Lirnnunfhes douglusii involved in triacylglycerol synthesis has an unusual specificity for very long chain acyl groups in both of its substrates, namely acyl-CoA and S I I -1 -acylglycerol-3-phosphate, and causes the enrichment of erucoyl groups in the sn-2 position of the seed oil of this plant species. We have isolated a cDNA clone encoding this embryo-specific, microsomal acyltransferase via heterologous complementation of an Escherichia coli mutant deficient in sn-1 -acylglycerol-3-phosphate acyltransferase activity. The open reading frame of the cDNA insert encodes a protein with a length of 281 amino acids, with three predicted membrane-spanning domains and of about 31.7 kDa. The sequence exhibits substantial sequence similaritiy to the sn-1 -acylglycerol-3-phosphate acyltransferase of E. coli. The corresponding transcript was detectable in developing embryos but not in leaves of L. douglasii, and expression of the open reading frame in E. coli caused sn-l-acylglycerol-3-phosphate acyltransferase activity which showed properties different from those of the bacterial acyltransferase but typical of the L. douglusii enzyme involved in triacylglycerol biosynthesis.

The erucoyl-CoA specific sn-1-acylglycerol-3-phosphate acyltransferase (LPAAT) of Limnanthes douglasii was functionally expressed in developing seeds of differing high-erucic acid rapeseed genotypes, namely resynthesized lines and cultivars. Lipid analysis revealed that seed oil of transgenic plants in contrast to that of control plants contained trierucoylglycerol as well as a molecular species with two erucoy1 groups and one eicosenoyl group. The proportion of trierucoylglycerol was distinctly higher in the seeds from transgenic resynthesized plants than in those from transgenic cultivars. In pooled seed oil fractions, up to 9% trierucoylglycerol (trierucin) was determined and the fatty acid composition at the sn-2 position was found to consist of more than 40% erucic acid. Since the pooled seeds were segregating for the presence of the L. douglasii gene, the analysis of single seeds gave even higher levels of up to 13% trierucin. Biosynthese von Trierucoylglycerol in transgener Rapssaat(Brassica napus L). Die erucoyl-CoA-spezifische sn-1-Acylglycerin-3-phosphat-Acyltransferase (LPAAT) aus Limnanthes douglasii wurde in den reifenden Samen verschiedener erucasaurereicher Rapssorten und Resyntheselinien funktional exprimiert. Wie Lipidanalysen zeigten, enthielt das Samenol transgener Pflanzen, im Unterschied zu den Kontrollen,Trierucin sowie eine molekulare Spezies mit zwei Erucasaure-und einem Eicosensaurerest. Im Vergleich der Sorten war bei den Resyntheselinien der Trierucinanteil in den Samen transgener Pflanzen deutlich hoher. In vereinigten Samenolfraktionen betrug der Trierucingehalt bis zu 9% und der Erucasauregehalt in der sn-2-Position mehr als 40%. Aufgrund der Segregation des L. douglasii-Gens konnten bei der Analyse einzelner Samen hohere Trierucingehalte von bis zu 13% gemessen werden.