Siroheme Biosynthesis in Higher Plants

Abstract

Siroheme, the prosthetic group for both nitrite and sulfite reductases, is a methylated, iron-containing modified tetrapyrrole. Here we report the first molecular characterization of the branch point enzyme in higher plants, which directs intermediates toward siroheme synthesis. A cDNA was cloned from Arabidopsis thaliana (UPM1) that functionally complements an Escherichia coli cysG mutant, a strain that is unable to catalyze the conversion of uroporphyrinogen III (Uro'gen-III) to siroheme. UPM1 is 1484 base pairs and encodes a 369-amino acid, 39.9-kDa protein. The UPM1 product contains two regions that are identical to consensus sequences found in bacterial Uro'gen-III and precorrin methyltransferases. Recombinant UPM1 protein was found to catalyze S-adenosyl-L-methionine-dependent transmethylation by UPM1 in a multistep process involving the formation of a covalently linked complex with S-adenosyl-L-methionine. The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids. The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria. Genomic blot analysis showed that UPM1 is encoded in the A. thaliana genome. The genomic DNA corresponding to UPM1 was cloned and sequenced and found to contain at least five introns.

Tetrapyrrole Metabolism inArabidopsis thaliana

“…In Arabidopsis, this reaction is catalyzed by an SAM-dependent Urogen III methyltransferase encoded by UPM1 (At5g40850) (Leustek et al 1997). UPM1 encodes a 39.9-kDa protein containing two regions that are identical to consensus sequences found in bacterial Urogen III and precorrin methyltransferases encoded by SirA.…”

Identification and Characterization of the Terminal Enzyme of Siroheme Biosynthesis from Arabidopsis thaliana

“…However, in other bacteria such as Bacillus megaterium, these activities are housed within three separate enzymes called SirA (uro'gen III methyltransferase), SirC (dihydrosirohydrochlorin dehydrogenase), and SirB (sirohydrochlorin ferrochelatase) (17). We had already shown that Arabidopsis contains a functional SirA orthologue UPM1 (14). Using the BLAST algorithm we were unable to identify orthologues in the Arabidopsis genome or in any plant EST data base of SirC.…”

“…The enzyme that catalyzes this reaction, uro'gen III methyltransferase (UPM1), requires S-adenosyl-L-methionine as a methyl donor and has been characterized from a wide variety of sources including eubacteria (10,11), Archaea (12), and higher plants (13,14). In Arabidopsis thaliana UPM1 has been localized to the chloroplast (14). To complete the synthesis of siroheme, dihydrosirohydrochlorin has to be oxidized to sirohydrochlorin and then chelated with ferrous iron.…”

Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

“…The results described here show that the biosynthesis of sirohaem can be accomplished with a two-enzyme system. Two-enzyme systems for sirohaem biosynthesis have been inferred from previous work on Bacillus megaterium [12] and in the higher plant Arabidopsis thaliana [35]. Recently sirohaem synthesis from uro'gen III in S. cere isiae has been shown to require two proteins, Met1 and Met8 [17], although the functions of the two proteins have not been accurately ascribed.…”

“…(a) Alignment of the predicted amino acid sequences of the uro'gen III transmethylase sequences (CysG A ) from Anacystis nidulans [42], Synechocystis sp. (accession number ss110378), Arabidopsis thaliana [35], Zea mays [43], Paracocccus denitrificans [44], Methanobacterium ivanovii [45], Methanococcus jannaschii [46], Bacillus subtilis [18], Bacillus megaterium [12], Bacillus stearothermophilus [47], Escherichia coli [13], Propioni freudenreichii [48] and Pseudomonas denitrificans [10]. The consensus sequence is shown and the mutated residues are marked by an asterisk.…”