Structure and expression of chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.) isolated by redundant polymerase chain reaction.
Porphobilinogen (PBG) deaminase catalyzes the polymerization of four PBG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. Degenerate oligonucleotide primers were designed based on amino acid sequence data (generated by mass spectrometry) for purified PBG deaminase from pea (Pisum sativum L.) chloroplasts. These primers were used in TaqI polymerase-catalyzed polymerase chain reaction (PCR) amplification to produce partial cDNA and nuclear genomic fragments encoding the enzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA library constructed in lambda gt11 from leaf poly(A)+ RNA with the PCR products. The cDNA encodes an approximately 40-kD polypeptide containing a 46-amino acid NH2-terminal transit peptide and a mature protein of 323 amino acids. The deduced amino acid sequence of the mature pea enzyme is similar to PBG deaminases from other species and contains the conserved arginine and cysteine residues previously implicated in catalysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tissues and is subject to light induction.
Witty
M
M
Department of Plant Sciences, University of Cambridge, United Kingdom.
Wallace-Cook
A D
AD
Albrecht
H
H
Spano
A J
AJ
Michel
H
H
Shabanowitz
J
J
Hunt
D F
DF
Timko
M P
MP
Smith
A G
AG
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002736:Chloroplasts / Q000201:enzymology*; D004247:DNA / Q000032:analysis / Q000737:chemistry; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D011163:Hydroxymethylbilane Synthase / Q000096:biosynthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D011163:Hydroxymethylbilane Synthase