SummaryMg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin iX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg-chelatase activity.In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide.

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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...

Background: Squamous cell carcinomas are common skin tumors in cats. We investigated photodynamic therapy (PDT) using a new liposomal photosensitizer as a minimally invasive, effective treatment that can be easily performed while achieving good cosmetic results.Aim: The goal of this study was to assess and describe possible toxicities using a liposomal formulation of the photosensitizer meta-(Tetrahydroxyphenyl)Chlorin (m-THPC) and investigate if favorable pharmacokinetics translate into favorable tumor response and control.Animals: Eighteen client-owned cats with 20 spontaneous cutaneous squamous cell carcinomas were included in the study. Methods: PDT was performed using a new, liposomal formulation of the photosensitizer. Toxicity, tumor response, and tumor control were evaluated retrospectively.Results: No general adverse effects were observed in cats treated with the new liposomal formulation. Mild local toxicity such as erythema and edema were seen in 15% of the patients. All cats responded to therapy, with a complete response rate of 100%. The overall 1-year control rate was 75%. The tumor recurrence rate was 20% with a median time to recurrence of 172.25 (687.1) days.Conclusions and Clinical Importance: A new liposomal photosensitizer was successfully used for squamous cell carcinoma in cats and was well tolerated. There were no systemic adverse effects observed with the liposomal formulation. The favorable pharmacokinetics of the liposomal drug resulted in a favorable tumor response.

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