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 P BG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. De generate oligonucleotide primers were designed based on amino acid seq uence 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) amplificatio n to produce partial cDNA and nuclear genomic fragments encoding the e nzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA li brary constructed in lambdagt11 from leaf poly(A)+ RNA with the PCR pr oducts. 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 enz yme is similar to PBG deaminases from other species and contains the c onserved arginine and cysteine residues previously implicated in catal ysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tis sues and is subject to light induction.