Siroheme biosynthesis in higher plants. Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana.
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.
Leustek
T
T
Center for Agricultural Molecular Biology and Plant Science Department, Rutgers University New Brunswick, New Jersey 08903-0231, USA. leustek@aesop.rutgers.edu
Smith
M
M
Murillo
M
M
Singh
D P
DP
Smith
A G
AG
Woodcock
S C
SC
Awan
S J
SJ
Warren
M J
MJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D017343:Genes, Plant; D006418:Heme / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006418:Heme; C009272:siroheme; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase