Crystal structure and substrate binding modeling of the uroporphyrinogen-III decarboxylase from Nicotiana tabacum - Implications for the catalytic mechanism
ABSTRACT The enzymatic catalysis of many biological processes of life is supported by the presence of cofactors and prosthetic groups originating from the common tetrapyrrole precursor uroporphyrinogen-III. Uroporphyrinogen-III decarboxylase catalyzes its conversion into coproporphyrinogen-III, leading in plants to chlorophyll and heme biosynthesis. Here we report the first crystal structure of a plant (Nicotiana tabacum) uroporphyrinogen-III decarboxylase, together with the molecular modeling of substrate binding in tobacco and human enzymes. Its structural comparison with the homologous human protein reveals a similar catalytic cleft with six invariant polar residues, Arg(32), Arg(36), Asp(82), Ser(214) (Thr in Escherichia coli), Tyr(159), and His(329) (tobacco numbering). The functional relationships obtained from the structural and modeling analyses of both enzymes allowed the proposal for a refined catalytic mechanism. Asp(82) and Tyr(159) seem to be the catalytic functional groups, whereas the other residues may serve in substrate recognition and binding, with Arg(32) steering its insertion. The crystallographic dimer appears to represent the protein dimer under physiological conditions. The dimeric arrangement offers a plausible mechanism at least for the first two (out of four) decarboxylation steps.
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ABSTRACT: Comparison of protein expression in necrotic leaves and in normal leaves of wheat (Triticum aestivum L.) showed that the abundance of 39 proteins was changed significantly, and 26 of these proteins were identified. Analysis of the function of the differentially expressed proteins in the necrotic hybrid leaves showed that the cytoprotective heat shock proteins may be induced to maintain the integrity of other proteins, facilitating the intercellular transportation of vital cellular enzymes upon necrosis. The increased abundance of NADH dehydrogenase indicated that the chloroplasts of necrotic leaves were under photo-oxidative stress. In addition, the light and dark events of photosynthesis were impacted differently during necrosis. The increased abundance of the hormone-sensitive enzymes phospholipase and β-1,3-glucanase suggested that the level of plant hormones may be increased in necrotic leaves. Both DNA helicase and maturase K were down-regulated in necrotic leaves, indicating basic genetic processes, including replication, repair, recombination, transcription and translation, were impacted during necrosis. The results of this study give a comprehensive picture of the post-transcriptional response to necrosis in hybrid wheat leaves and serve as a platform for further characterization of gene function and regulation in wheat hybrid necrosis.Plant Science 09/2008; DOI:10.1016/j.plantsci.2008.05.017 · 4.11 Impact Factor
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ABSTRACT: Uroporphyrinogen decarboxylase (UroD) (EC 126.96.36.199) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga (Chlorella). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23-0.3 mg protein/mL, approximately 6-8 micromol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.Canadian Journal of Microbiology 03/2007; 53(2):303-12. DOI:10.1139/W06-117 · 1.18 Impact Factor
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ABSTRACT: Cloning, sequencing and functional analysis of the Kluyveromyces lactis KlHEM12 gene and its upstream region are reported. The gene encodes for a protein that is highly homologous to uroporphyrinogen decarboxylases from different organisms and complements its mutation in Saccharomyces cerevisiae. Secondary structure prediction allows outlining a topology diagram which is compatible with a (beta/alpha)8-barrel structure. A K. lactis haploid strain carrying a null allele of KlHEM12 showed decreased growth in media not supplemented with hemin (ferriprotoporphyrin IX) and red-fluorescent colonies due to the accumulation of porphyrins. KlHEM12 expression was analysed by Northern blot and promoter fusion to the reporter lacZ gene. Transcription of this gene is not under heme or glucose repression and it is slightly induced by non-fermentable carbon sources through the Hap2/3/4/5 complex.Current Genetics 10/2004; 46(3):147-57. DOI:10.1007/s00294-004-0517-5 · 1.71 Impact Factor