Biochimica et Biophysiea Acre, ]0760991) 29-36
© 1991ElsevierSci~mc~PublishersB.V.(BiomedicalDivision)0167-4838/91/$03,50 ADONIS 0|67483891000620
29
BBAPRO33791
Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea ( Pisum satioum L.) Anthony J. Spano and Michael P. Timko Dtpartment o/Biology. Universityof Virginia. Chadotte~ville. 7A fU.S.A.)
(Received27June1990} Keywords: porphobilinogcndeamin~e;Chloroplast:Cltlotophyllbiosynthesis;(P~sum) Porphobilinogen dearninase catalyzes the condensation of four porphobilinogen monopytrole units into hydroxymelhylbilane, a linear tetrapynele necessary for the formation of cldurophyll and heine in hillher plant cells. We report file puxilieation to homogeneity of a cblorOl~-'t.localized form o| the enzyme from pea (Pisum ~ t i v u m L) by a novel pmilicafion scheme incolvinlgdye-ligend aff'mity chromatography. The pufilied chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by slze-excluslon chromatography and sodium ~deeyl sulfate polyacfflamide gel electrephoresis, The isoeleetrlc point of the protein is acidic. The activity of the enzyme shows dllferant levels o! sensitivity to divalent cations and is most sensitive to Fe z4". The amino terminus of pea enzyme has been oblained by microsequeneing and determined to bear little similarity to the amino acid sequences of pu~pbubilinogendeaminases purified from other organisms. Polyclonal anti~ra elicited against the purified proiein has been used to examine the abumlance and cellular distribution of the enzyme. Introduction Tetrapyrroles and their derivatives, notably the homes, sirohemes and chlorophylls, play an important role in the growth and differentiation of higher plant cells. Home is present in all cell types, where it functions as the prosthetic group of integral membrane components (e.g., cytochromes of plastidic and mitochondtial electron transport chains) as well as numerous soluble proteins (e.g., catalase, pvroxidase, nitrite and sulfite reductases). In contrast, the chlorophylls are restrictvd to the chloroplast thylakoid membranes in photosynthetic cells where they serve as chromophores in the Photosystem I and Photosystem II reaction centers and light-harvesting apparatus. In all organisms studied thus far the first committed step in tetrapyrrole formation is the synthesis of 8aminolevulinic acid (ALA) [1,2]. In some prokatyotes and most eukaryotes the formation of ALA is mediated
Abbv.-.viations:ALA, 8-aminolevaliaicacid; SDS.PAGE,~tium dodecyl sulphate polyacrylaroide gel electrophoresis" PBG, porphobilir~ogen. Correspondence:M.P.Timk¢~Departmentof Biology,University of Virginia,Charlottesville,VA 22901,U.S.A.
by the soluble enzyme ALA synthase, in yeast and animal cells, ALA synthase has been demonstrated to be a nuckar-encoded, mitochondria-localized activity [1,2]. In higher plants and green algae the formation of ALA is mediated by a series of plastid-localized en. zymatic activities involving novel tRNA intermediates [1]. Two molecules of ALA are subsequently converted imo one mol~ule of porphobilinogen (PBG) by the activity of a porphobilinogen synthasc. Four monopyr. role PBG units are then condensed into a linear tetra, pyrrole, termed hydroxymethylbilane, by the enzyme porphobifinogen deaminase (hydroxymethylbilane syn* thase, EC 4.3.1.8) [3]. Hydroxymethylbilane is cyclizcd and isomerized to yield uroporphorinogen IIl, the precursor of all biologically active homes and chlorophylls. The biochemical and kinetic properties of porphobb [inogen deaminases from a variety of prokaryotic and eukaryotic organisms have been reported [4,27,5]. The exact sequence of steps involved in the assembly of the linear hydroxymethylbilane on the porphobilinogen deaminase backbone is also known [6-8]. Additionally, it has now been demonstrated that the assembly of hydroxymethylbilane on the porphobilinogen deaminase backbone involves a dipyrromethane ~factor [7,8]. Cloned cDNAs or ganomic sequences encoding porphobllinogen deaminase have been isolated and characterized from a variety of organisms including E.
30
cell [9], Eugtena [10], yeast [11] and animal cells [1233]. As a consequence of its involvement in certain genetic porphyrias, considerable effort has been devoted toward understanding the molecular genetic mechanisms active in coatroll;,ng the synthesis and activity of porphobilinogen deaminas¢ in mammalian cells {13]. Little informatioo is presently available on the synthesis and regulation of ~!his protein in higher plants. it has been previously suggested that duplicate pathways for tctrapyrrole synthesis exist in higher plant cells, namely ~ chloroplast-localized pathway for the synthesis of chlorophyll and plastid heme and an extraplastidic patbway for heroe synthesis [14,15]. The extent of this duplication and its significance are unknown. These dual pathways, however, must be coordinated and integrated since they share a common pool of biosynthetic intermediates [2]. As a step toward understanding the molecular genetic control of tetrapyrrote formatioo in plant cells, we have examined the chloroplast-localized porphobiiinogen deaminase activity from pea, (Pisum sativum L.). We describe a novel and efficient procedure for the purifi. cation of porphobilinogen deaminase from plant tissue. The properties of the purified plastid-localized enzyme and its developmental and cell.specific distribution are discussed. Materials e~l Methods
P~ant materials and growth Pea seeds (Pisum sativum L. vat. Progress No. 9) (obtained from Burpee Seed Company) were imbibed in distilled water overnight at room temperature and planted in moistened vermiculite. Plants used for enzyme isolation were grown at 28°C undr 16 h of illumination for 10-14 days prior to harvesting of leaves. Plants used in greening studies were grown in total darkness at 28
© 1991ElsevierSci~mc~PublishersB.V.(BiomedicalDivision)0167-4838/91/$03,50 ADONIS 0|67483891000620
29
BBAPRO33791
Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea ( Pisum satioum L.) Anthony J. Spano and Michael P. Timko Dtpartment o/Biology. Universityof Virginia. Chadotte~ville. 7A fU.S.A.)
(Received27June1990} Keywords: porphobilinogcndeamin~e;Chloroplast:Cltlotophyllbiosynthesis;(P~sum) Porphobilinogen dearninase catalyzes the condensation of four porphobilinogen monopytrole units into hydroxymelhylbilane, a linear tetrapynele necessary for the formation of cldurophyll and heine in hillher plant cells. We report file puxilieation to homogeneity of a cblorOl~-'t.localized form o| the enzyme from pea (Pisum ~ t i v u m L) by a novel pmilicafion scheme incolvinlgdye-ligend aff'mity chromatography. The pufilied chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by slze-excluslon chromatography and sodium ~deeyl sulfate polyacfflamide gel electrephoresis, The isoeleetrlc point of the protein is acidic. The activity of the enzyme shows dllferant levels o! sensitivity to divalent cations and is most sensitive to Fe z4". The amino terminus of pea enzyme has been oblained by microsequeneing and determined to bear little similarity to the amino acid sequences of pu~pbubilinogendeaminases purified from other organisms. Polyclonal anti~ra elicited against the purified proiein has been used to examine the abumlance and cellular distribution of the enzyme. Introduction Tetrapyrroles and their derivatives, notably the homes, sirohemes and chlorophylls, play an important role in the growth and differentiation of higher plant cells. Home is present in all cell types, where it functions as the prosthetic group of integral membrane components (e.g., cytochromes of plastidic and mitochondtial electron transport chains) as well as numerous soluble proteins (e.g., catalase, pvroxidase, nitrite and sulfite reductases). In contrast, the chlorophylls are restrictvd to the chloroplast thylakoid membranes in photosynthetic cells where they serve as chromophores in the Photosystem I and Photosystem II reaction centers and light-harvesting apparatus. In all organisms studied thus far the first committed step in tetrapyrrole formation is the synthesis of 8aminolevulinic acid (ALA) [1,2]. In some prokatyotes and most eukaryotes the formation of ALA is mediated
Abbv.-.viations:ALA, 8-aminolevaliaicacid; SDS.PAGE,~tium dodecyl sulphate polyacrylaroide gel electrophoresis" PBG, porphobilir~ogen. Correspondence:M.P.Timk¢~Departmentof Biology,University of Virginia,Charlottesville,VA 22901,U.S.A.
by the soluble enzyme ALA synthase, in yeast and animal cells, ALA synthase has been demonstrated to be a nuckar-encoded, mitochondria-localized activity [1,2]. In higher plants and green algae the formation of ALA is mediated by a series of plastid-localized en. zymatic activities involving novel tRNA intermediates [1]. Two molecules of ALA are subsequently converted imo one mol~ule of porphobilinogen (PBG) by the activity of a porphobilinogen synthasc. Four monopyr. role PBG units are then condensed into a linear tetra, pyrrole, termed hydroxymethylbilane, by the enzyme porphobifinogen deaminase (hydroxymethylbilane syn* thase, EC 4.3.1.8) [3]. Hydroxymethylbilane is cyclizcd and isomerized to yield uroporphorinogen IIl, the precursor of all biologically active homes and chlorophylls. The biochemical and kinetic properties of porphobb [inogen deaminases from a variety of prokaryotic and eukaryotic organisms have been reported [4,27,5]. The exact sequence of steps involved in the assembly of the linear hydroxymethylbilane on the porphobilinogen deaminase backbone is also known [6-8]. Additionally, it has now been demonstrated that the assembly of hydroxymethylbilane on the porphobilinogen deaminase backbone involves a dipyrromethane ~factor [7,8]. Cloned cDNAs or ganomic sequences encoding porphobllinogen deaminase have been isolated and characterized from a variety of organisms including E.
30
cell [9], Eugtena [10], yeast [11] and animal cells [1233]. As a consequence of its involvement in certain genetic porphyrias, considerable effort has been devoted toward understanding the molecular genetic mechanisms active in coatroll;,ng the synthesis and activity of porphobilinogen deaminas¢ in mammalian cells {13]. Little informatioo is presently available on the synthesis and regulation of ~!his protein in higher plants. it has been previously suggested that duplicate pathways for tctrapyrrole synthesis exist in higher plant cells, namely ~ chloroplast-localized pathway for the synthesis of chlorophyll and plastid heme and an extraplastidic patbway for heroe synthesis [14,15]. The extent of this duplication and its significance are unknown. These dual pathways, however, must be coordinated and integrated since they share a common pool of biosynthetic intermediates [2]. As a step toward understanding the molecular genetic control of tetrapyrrote formatioo in plant cells, we have examined the chloroplast-localized porphobiiinogen deaminase activity from pea, (Pisum sativum L.). We describe a novel and efficient procedure for the purifi. cation of porphobilinogen deaminase from plant tissue. The properties of the purified plastid-localized enzyme and its developmental and cell.specific distribution are discussed. Materials e~l Methods
P~ant materials and growth Pea seeds (Pisum sativum L. vat. Progress No. 9) (obtained from Burpee Seed Company) were imbibed in distilled water overnight at room temperature and planted in moistened vermiculite. Plants used for enzyme isolation were grown at 28°C undr 16 h of illumination for 10-14 days prior to harvesting of leaves. Plants used in greening studies were grown in total darkness at 28
