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 pyridoxal 5'-phosphate-dependent enzyme cystathionine beta-lyase (CBL) catalyzes the penultimate step in the de novo biosynthesis of Met in microbes and plants. Absence of CBL in higher organisms makes it an important target for the development of antibiotics and herbicides. The three-dimensional structure of cystathionine beta-lyase from Arabidopsis was determined by Patterson search techniques, using the structure of tobacco (Nicotiana tabacum) cystathionine gamma-synthase as starting point. At a resolution of 2.3 A, the model was refined to a final crystallographic R-factor of 24.9%. The overall structure is very similar to other pyridoxal 5'-phosphate-dependent enzymes of the gamma-family. Exchange of a few critical residues within the active site causes the different substrate preferences between Escherichia coli and Arabidopsis CBL. Loss of interactions at the alpha-carboxyl site is the reason for the poorer substrate binding of Arabidopsis CBL. In addition, the binding pocket of Arabidopsis CBL is larger than that of E. coli CBL, explaining the similar binding of L-cystathionine and L-djenkolate in Arabidopsis CBL in contrast to E. coli CBL, where the substrate binding site is optimized for the natural substrate cystathionine.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 Å resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open ␣/␤ architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the ␤-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.Threonine synthase (TS, EC 4.2.99.2) 1 is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme that catalyzes the ultimate step in threonine biosynthesis, the PLP-dependent ␤,␥-replacement reaction (Reaction 1) of O-phosphohomoserine (OPHS) yielding threonine and inorganic phosphate (1-3).Together with tryptophan synthase (TRPS), threonine deaminase (TDA), O-acetylserine sulfhydrylase (OASS), cystathione ␤-synthase (CBS), and 1-aminocyclopropane-1-carboxylate deaminase (ACCD), TS constitutes the core of the fold-type II family of PLP enzymes (4, 5) (also referred to as ␤-family (6)). Detailed amino acid sequence alignments revealed that TS can be grouped into a plant and a fungal subfamily (7). The former one (class I subfamily) comprises TS from higher plants, cyanobacteria, archaebacteria, and the eubacterial groups of Mycobacteria, Aquificaceae, and Bacillus species. The second subfamily (class II subfamily) contains the enzymes from fungi and from the eubacterial groups of Proteobacteria and corneyform bacteria. Only 5 from ϳ500 residues are invariant between both subfamilies, including the PLP-binding lysine as part of a phenylalanine-lysine-aspartate consensus sequence.During the last decades, TS was purified and characterized from several bacteria and fungi (8 -12) and from Arabidopsis thaliana (7, 13). In plants, the substrate of TS, OPHS, is the branching point for threonine and methionine biosynthesis. Flux coordination between both synthetic pathways is accomplished by allosteric activation of plant threonine synthase. The allosteric effector is S-adenosyl methionine (SAM) (14, 15), a product of methioni...

Ribosomal protein L7/L12, the only multicopy component of the ribosome, is involved in translation factor binding and in the ribosomal GTPase center. The gene for L7/L12 from Thermotoga maritima was cloned and the protein expressed at high levels in Escherichia coli. Purification of L7/L12 was achieved under non-denaturing conditions via heat treatment and two chromatographic steps. Circular dichroism melting profiles were monitored at 222 nm, showing the melting temperature of the protein at pH 7.5 around 110 degrees C, compared to approximately 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely followed the path of the thermal melting. Dynamic light scattering, gel filtration chromatography, and crosslinking experiments suggested that under physiological buffer conditions Thermotoga maritima L7/L12 exists as a tetramer. The protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (12(1)3) space group with an estimated two and three to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed which improved the mosaic spreads and the resolution limits. For the structure solution isoleucine was mutated to methionine at two separate positions, the mutant forms expressed as selenomethionine variants and crystallized.

No abstract