PLANT PHYSIOLOGY , Vol 103, Issue 1 139-147, Copyright © 1993 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction
M. Witty, ADM. Wallace-Cook, H. Albrecht, A. J. Spano, H. Michel, J. Shabanowitz, D. F. Hunt, M. P. Timko and A. G. Smith
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA United Kingdom (M.W., A.D.M.W.-C., H.A., A.G.S.)
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 Taql
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.
