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.
Chapter: Hemes in BiologyWiley Encyclopedia of Chemical Biology, 05/2008; , ISBN: 9780470048672
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ABSTRACT: The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. They contain a characteristic sequence motif which is termed an LHC motif consisting of 25-30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs which are termed LILs. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein was previously shown to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane (TM) domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the TM domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (E171, N174 and D189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol biosynthesizing activity, however, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.Journal of Biological Chemistry 11/2013; 289(2). DOI:10.1074/jbc.M113.525428 · 4.60 Impact Factor
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ABSTRACT: The purple bacteria make bacteriochlorophylls for the photosynthetic mode of growth. These pigments are made from the simple precursors glycine and succinyl CoA and the initial steps in the pathway of bacteriochlorophyll biosynthesis are shared with the vitamin B12 and heme biosynthetic pathways. This chapter concentrates on the biochemical properties of the enzymes involved in each step of the pathway and the discovery and assignment of the genes encoding these enzymes. The characterization of purple bacterial enzymes involved in these steps has been crucial in understanding similar enzymes from other sources. The characterization of the early steps in the pathway within purple bacteria, such as δ-aminolevulinate synthase, contributed significantly to the understanding of the mammalian enzymes in the 1950s and 1960s. More recently the study of the purple bacterial enzymes toward the end of the pathway has been instrumental in identifying and characterizing the orthologous enzymes from cyanobacteria and plants. In this review we present the details of the properties of these enzymes from the purple bacteria, such as purification methods and kinetic analyses from the early literature, through to more recent studies using recombinant purple bacterial enzymes.12/2007: pages 57-79;