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The Role of the Subunit CHL D in the Chelation Step of Protoporphyrin IX

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The Role of the Subunit CHL D in the Chelation Step of Protoporphyrin IX Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1941–1946, March 1999 Biochemistry Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX SUSANNA GRA¨FE*, HANS-PETER SALUZ*, BERNHARD GRIMM†, AND FRANK HA¨NEL*‡ *Hans-Kno¨ll-Institut fu¨r Naturstoff-Forschung e.V., Department of Cell and Molecular Biology, Beutenbergstr. 11, 07745 Jena, Germany; and †Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung, AG Chlorophyll Biosynthesis, Corrensstr. 3, 06466 Gatersleben, Germany Edited by Lawrence Bogorad, Harvard University, Cambridge, MA, and approved December 21, 1998 (received for review July 20, 1998) ABSTRACT The Mg-chelation is found to be a prerequisite CHL I, and CHL H proteins from tobacco in yeast and measuring to direct protoporphyrin IX into the chlorophyll (Chl)- the activity of the recombinant enzyme complex in yeast extracts synthesizing branch of the tetrapyrrol pathway. The ATP- (11). dependent insertion of magnesium into protoporphyrin IX is The enzymatic insertion of magnesium into protoporphyrin IX catalyzed by the enzyme Mg-chelatase, which consists of three is catalyzed in two steps (12). First, an ATP-dependent activation protein subunits (CHL D, CHL I, and CHL H). We have chosen complex is formed that consists of the subunits CHL D (Bch D) the Mg-chelatase from tobacco to obtain more information about and CHL I (Bch I). When tested individually Bch I and Bch D had the mode of molecular action of this complex enzyme by eluci- no ATPase activity, but when combined they hydrolyzed ATP (6, dating the interactions in vitro and in vivo between the central 13). In addition, the physical interaction between CHL D and subunit CHL D and subunits CHL I and CHL H. We dissected CHL I as well as the homodimerization of CHL D were proven CHL D in defined peptide fragments and assayed for the by the yeast two-hybrid system (11). In a second ATP hydrolyzing essential part of CHL D for protein–protein interaction and step, Mg21 is inserted into protoporphyrin IX, which is most likely enzyme activity. Surprisingly, only a small part of CHL D, i.e., bound to CHL H (Bch H) (6). 110 aa, was required for interaction with the partner subunits The aim of this work was the elucidation of the enzymatic and and maintenance of the enzyme activity. In addition, it could be interactive domains of CHL D. This subunit is known to be highly demonstrated that CHL D is capable of forming homodimers. interesting because of its unusual structure (4, 11). The amino Moreover, it interacted with both CHL I and CHL H. Our data terminal half of CHL D shows significant similarity (46%) to the led to the outline of a two-step model based on the cooperation entire CHL I peptide sequence, indicating a duplication from an of the subunits for the chelation process. ancestral gene. The carboxyl-terminal moiety does not resemble any other known protein structure. Both parts of CHL D are Chelatases catalyze the insertion of divalent metal ions into linked by a glutamineyasparagineyproline-rich region, flanked by tetrapyrroles. The multiple tetrapyrrole endproducts contain a highly acid-rich domain of more than 26 aa residues. In various metal cations, such as Mg(II), Fe(II), Co(II), Ni(II), continuation of our previous work on the functional analysis of Zn(II), Mn(II), and Cu(II), which are inserted by chelatases. The the Mg-chelatase subunits, we dissected CHL D and defined the detailed mechanism of these enzymes is still not clear. Best known essential regions for the interaction with the other subunits that are the Mg and Fe chelatases (1). The Mg-chelatase is found to were required for the enzymatic activity. For the functional be specific for photosynthetic organisms. It catalyzes the insertion investigations we assayed the recombinant subunits expressed in of the magnesium ion into protoporphyrin IX, the last common yeast. It is well known that enzymes of higher eukaryotic organ- intermediate precursor in chlorophyll (Chl) and heme biosynthe- isms are usually functional when expressed in yeast systems and sis. The enzyme Fe chelatase plays an essential role in heme that for this kind of study bacterial organisms are less suited (14). synthesis by catalyzing the insertion of a ferrous ion into the We established a functional model for the assembly of the tetrapyrrole ring structure. Both of these enzymes use the same interacting subunits needed for the active Mg-chelatase complex. substrate, protoporphyrin IX (1). Despite this fact, the structure of these two proteins is entirely different. While the Mg-chelatase MATERIALS AND METHODS consists of three protein subunits, the ferrochelatase consists of Bacterial and Yeast Strains. Eschericha coli TG1[supE D only one protein (2). (hsdM-mrcB) 5 thi D(lac-proAB)F9 (tra D36pro AB1 laclq lacZ In bacteriochlorophyll (Bch)-synthesizing Rhodobacter spha- DM15)] (New England Biolabs) hosted all plasmids. E. coli BL21 eroides and Chlorobium vibrioforme as well as in the chlorophyll [F-dcm ompT hsdS(r m )gal] (Stratagene) was used for expres- y B- B- a-synthesizing cyanobacterium Synechocystis, the ORFs bchD sion of the glutathione S-transferase (GST)-CHLD(M LM ) y y 1 2 chlD, bchH chlH, and bchI chlI encode the proteins that are fusion protein (Table 1, no. 3). All other GST fusion proteins required for the reconstruction of in vitro Mg-chelatase activity were expressed in yeast strain CEN.PK2 (MATaya, ura3–52y (3–5). The proteins Bch H and Bch I of R. sphaeroides were ura3y52, trp1–289ytrp1–289, leu2–3yleu2–3, 112, his3–1yhis3–1) purified to homogeneity, and the protein Bch D was partially (a kind gift of K.D. Entian, University of Frankfurt, Germany). purified (6). It was shown that a monomer of H and a dimer of Two-hybrid experiments and reconstitution assays of Mg- I gave optimal Mg-chelatase activity (6). Furthermore, Xantha-f, chelatase activity in yeast were performed in yeast strains SFY526 -g, and -h have been shown to be the structural genes for the three [MATa, ura3–52, his3–200, ade2–101, lys2–801, trp1–901, leu2–3, Mg-chelatase subunits in barley (7, 8). The plant coding se- r 112-can , gal4–542, gal80–538, URA3::GAL1UAS-GAL1TATA- quences homologous to chlI and chlH were cloned several years ago (9, 10). Recently, we identified a sequence homologous to the This paper was submitted directly (Track II) to the Proceedings office. bacterial bchD gene and confirmed structural similarity between Abbreviations: Chl, chlorophyll; Bch, bacteriochlorophyll; GST, glu- the bacterial and plant Mg-chelatase by expressing the CHL D, tathione S-transferase; N, N terminal; NM1, N terminal and motif; NM1LM2, N terminal, motif, linker region, and motif; M1LM2, motif, The publication costs of this article were defrayed in part by page charge linker region and motif; M1LM2C, motif, linker region, motif, and C terminal; M C, motif and C terminal; C, C terminal; AD, activating payment. This article must therefore be hereby marked ‘‘advertisement’’ in 2 domain; BD, binding domain. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. e-mail: fhaenel@ PNAS is available online at www.pnas.org. pmail.hki-jena.de. 1941 Downloaded by guest on September 28, 2021 1942 Biochemistry: Gra¨fe et al. Proc. Natl. Acad. Sci. USA 96 (1999) Table 1. The recombinant plasmid constructions used in this work CHL D No. Construct Plasmid fragment Cloning sites 1 GST-CHL D(N) pEGKT 63-374 BamHIyHindIII 2 GST-CHL D(NM1LM2) pEGKT 63-485 BamHIyHindIII 3 GST-CHL D(M1LM2) pGEX4T-1 375-485 BamHIyEcoRI 4 GST-CHL D(M1LM2C) pEGKT 375-748 XbaIySalI 5 CHL D pGEM-7Zf(2) 63-748 XbalyBamHI 6 CHL I pGEM-7Zf(2) ChlI XbaIyHindIII 7 CHL H pCR II ChlH — 8 BD-CHL D(N) pAS2-1 63-374 EcoRIyBamHI 9 BD-CHL D(NM1) pAS2-1 63-398 EcoRIyBamHI 10 AD-CHL D(NM1) pACT2 63-398 BamHIyEcoRI 11 BD-CHL D(NM1LM2) pAS2-1 63-485 EcoRIyBamHI 12 AD-CHL D(NM1LM2) pACT2 63-485 BamHIyEcoRI 13 BD-CHL D(M1LM2) pAS2-1 375-485 EcoRIyBamHI 14 AD-CHL D(M1LM2) pACT2 375-485 BamHIyEcoRI 15 BD-CHL D(M1LM2) pACT2-1 375-748 EcoRIyBamHI 16 AD-CHL D(M1LM2C) pACT2 375-748 BamHIyEcoRI 17 BD-CHL D(M2C) pAS2-1 448-748 EcoRIyBamHI 18 AD-CHL D(M2C) pACT2 448-748 BamHIyEcoRI 19 BD-CHL D(C) pAS2-1 486-748 EcoRIyBamHI 20 (M1LM2C)CHL D-lexA pFBL23 375-748 EcoRIyBamHI 21 BD-CHL I-CHL D(M1LM2C) pAS2-1 375-748 BamHIySalI 22 CHL I p423TEF ChlI SmaIySalI 23 AD-CHL D pACT2 63-748 SmaIyEcoRI 24 AD-CHL I pACT2 ChlI SmaIyXhoI 25 AD-CHL H pACT2 ChlH SmaIyXhoI 26 BD-CHL D pAS2 63-748 SmaIySalI 27 BD-CHL I pAS2 ChlI SmaIySalI 28 BD-CHL H pAS2 ChlH SmaIySalI lacZ] (CLONTECH) and L40 [MATa, his3D200, trp1–901, CGT GGG AG-39;59-AGA TAG GAT CCA CCT CTA TCT 9 9 leu2–3, 112, ade2, LYS2::(lexAop)4-HIS3, URA3::(lexAop)8- TCG GAA AAA GAT G-3 ;5-AGA TAG GAT CCA AGC lacZ)] (a kind gift of S. Hollenberg, Fred Hutchinson Cancer TTT CCA TCG ATG GCA GCT AAG CA-39;59-AGA TAG Research Center, Seattle, WA). GAT CCG AAT TCA TCT TTT CCG AAG ATA GAG GT-39; Construction of Fusion Proteins Between CHL D Fragments 59-AGA TAG AAT TCA TGG TGG AAC CTG AAA AAC and GAL4 Binding andyor Activation Domains or LexA-DNA AAC C-39;59-AGA TAG AAT TCA TGG ATG CGG AAG Binding Domain.
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