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1.
Sawers RJ Viney J Farmer PR Bussey RR Olsefski G Anufrikova K Hunter CN Brutnell TP 《Plant molecular biology》2006,60(1):95-106
Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll
biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium
chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function.
Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified
in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition
of wild-type SsChlI. 相似文献
2.
Vanessa Lake Ulf Olsson Robert D Willows Mats Hansson 《European journal of biochemistry》2004,271(11):2182-2188
During biosynthesis of chlorophyll, Mg(2+) is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximately 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called "ATPases associated with various cellular activities", known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg(2+) and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis. 相似文献
3.
Joakim Lundqvist Ilka Braumann Marzena Kurowska André H. Müller Mats Hansson 《The Journal of biological chemistry》2013,288(33):24012-24019
The ATP-dependent insertion of Mg2+ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg2+ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity. 相似文献
4.
The Magnesium-Chelatase H Subunit Binds Abscisic Acid and Functions in Abscisic Acid Signaling: New Evidence in Arabidopsis 总被引:1,自引:0,他引:1
Fu-Qing Wu Qi Xin Zheng Cao Zhi-Qiang Liu Shu-Yuan Du Chao Mei Chen-Xi Zhao Xiao-Fang Wang Yi Shang Tao Jiang Xiao-Feng Zhang Lu Yan Rui Zhao Zi-Ning Cui Rui Liu Hai-Li Sun Xin-Ling Yang Zhen Su Da-Peng Zhang 《Plant physiology》2009,150(4):1940-1954
Using a newly developed abscisic acid (ABA)-affinity chromatography technique, we showed that the magnesium-chelatase H subunit ABAR/CHLH (for putative abscisic acid receptor/chelatase H subunit) specifically binds ABA through the C-terminal half but not the N-terminal half. A set of potential agonists/antagonists to ABA, including 2-trans,4-trans-ABA, gibberellin, cytokinin-like regulator 6-benzylaminopurine, auxin indole-3-acetic acid, auxin-like substance naphthalene acetic acid, and jasmonic acid methyl ester, did not bind ABAR/CHLH. A C-terminal C370 truncated ABAR with 369 amino acid residues (631–999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently, expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescent protein (GFP) in wild-type plants conferred ABA hypersensitivity in all major ABA responses, including seed germination, postgermination growth, and stomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cch mutant of the ABAR/CHLH gene restored the ABA sensitivity of the mutant in all of the ABA responses. However, the effect of expression of the ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effect of the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles of ABAR/CHLH from the mutant pool in the Arabidopsis Biological Resource Center via Arabidopsis (Arabidopsis thaliana) Targeting-Induced Local Lesions in Genomes. The abar-2 mutant has a point mutation resulting in the N-terminal Leu-348→Phe, and the abar-3 mutant has a point mutation resulting in the N-terminal Ser-183→Phe. The two mutants show altered ABA-related phenotypes in seed germination and postgermination growth but not in stomatal movement. These findings support the idea that ABAR/CHLH is an ABA receptor and reveal that the C-terminal half of ABAR/CHLH plays a central role in ABA signaling, which is consistent with its ABA-binding ability, but the N-terminal half is also functionally required, likely through a regulatory action on the C-terminal half. 相似文献
5.
The Mg-Chelatase H Subunit of Arabidopsis Antagonizes a Group of WRKY Transcription Repressors to Relieve ABA-Responsive Genes of Inhibition 总被引:4,自引:0,他引:4
Yi Shang Lu Yan Zhi-Qiang Liu Zheng Cao Chao Mei Qi Xin Fu-Qing Wu Xiao-Fang Wang Shu-Yuan Du Tao Jiang Xiao-Feng Zhang Rui Zhao Hai-Li Sun Rui Liu Yong-Tao Yu Da-Peng Zhang 《The Plant cell》2010,22(6):1909-1935
6.
C. Gamini Kannangara Ute C. Vothknecht Mats Hansson Diter von Wettstein 《Molecular genetics and genomics : MGG》1997,254(1):85-92
Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272?000?×?g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively. 相似文献
7.
E. Chekounova V. Voronetskaya J. Papenbrock B. Grimm C. Beck 《Molecular genetics and genomics : MGG》2001,266(3):363-373
Two chlorophyll-deficient mutants of Chlamydomonas reinhardtii, chl1 and brs-1, are light sensitive and, when grown heterotrophically in the dark, accumulate protoporphyrin IX and exhibit yellow/orange pigmentation. The lesions in both mutants were mapped to the gene (CHLH) for the plastid-localized H subunit of the heterotrimeric magnesium chelatase that catalyzes the insertion of magnesium into protoporphyrin IX. The genetic defects in the mutants could be assigned to +1 frameshift mutations in exon 9 (chl1) and exon 10 (brs-1) of the CHLH gene. In both mutants, the H subunit of magnesium chelatase was undetectable, but, as shown for chl1, the steady-state levels of the I and D subunits were unaltered in comparison to wild type. The CHLH gene exhibits marked light inducibility: levels of both the mRNA and the protein product are strongly increased when cultures are shifted from from the dark into the light, suggesting that this protein may play a crucial role in the light regulation of chlorophyll biosynthesis. 相似文献
8.
Localization of Mg-Chelatase and Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Activities within Isolated, Developing Cucumber Chloroplasts 总被引:9,自引:6,他引:3
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Magnesium chelatase and magnesium protoporphyrin IX monomethyl ester (oxidative) cyclase activities were both sensitive to inhibition by p-chloromercuribenzoate in intact, developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts. Magnesium chelatase was also sensitive to the membrane-impermeable mercurial p-chloromercuribenzene sulfonate (PCMBS), while cyclase activity was only slightly sensitive. When the plastids were pretreated with PCMBS, triosephosphate dehydrogenase activity was inhibited very slightly, indicating that PCMBS does not readily penetrate through the chloroplast envelope. These results suggest that magnesium chelatase is located in the chloroplast envelope, while the cyclase is located deeper within the chloroplast. 相似文献
9.
10.
Magnesium chelatase is an AAA+ ATPase that catalyzes the first step in chlorophyll biosynthesis, the energetically unfavorable insertion of a magnesium ion into a porphyrin ring. This enzyme contains two AAA+ domains, one active in the ChlI protein and one inactive in the ChlD protein. Using a series of mutants in the AAA+ domain of ChlD, we show that this site is essential for magnesium chelation and allosterically regulates Mg2+ and MgATP2− binding. 相似文献
11.
12.
13.
Gibberellin and abscisic acid signalling in aleurone 总被引:1,自引:0,他引:1
14.
15.
Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis 总被引:2,自引:0,他引:2
Magnesium (Mg) chelatase is a heterotrimeric enzyme complex that catalyzes a key regulatory and enzymatic reaction in chlorophyll biosynthesis, the insertion of Mg(2+) into protoporphyrin IX. Studies of the enzyme complex reconstituted in vitro have shown that all three of its subunits, CHL I, CHL D, and CHL H, are required for enzymatic activity. However, a new T-DNA knockout mutant of the chlorina locus, ch42-3 (Chl I), in Arabidopsis is still able to accumulate some chlorophyll despite the absence of Chl I mRNA and protein. In barley (Hordeum vulgare), CHL I is encoded by a single gene. We have identified an open reading frame that apparently encodes a second Chl I gene, Chl I2. Chl I1 and Chl I2 mRNA accumulate to similar levels in wild type, yet CHL I2 protein is not detectable in wild type or ch42-3, although the protein is translated and stromally processed as shown by in vivo pulse labeling and in vitro chloroplast imports. It is surprising that CHL D accumulates to wild-type levels in ch42-3, which is in contrast to reports that CHL D is unstable in CHL I-deficient backgrounds of barley. Our results show that limited Mg chelatase activity and CHL D accumulation can occur without detectable CHL I, despite its obligate requirement in vitro and its proposed chaperone-like stabilization and activation of CHL D. Thus, the unusual post-translational regulation of the CHL I2 protein provides an opportunity to study the different steps involved in stabilization and activation of the heterotrimeric Mg chelatase in vivo. 相似文献
16.
17.
Ikegami A Yoshimura N Motohashi K Takahashi S Romano PG Hisabori T Takamiya K Masuda T 《The Journal of biological chemistry》2007,282(27):19282-19291
Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370-375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts. 相似文献
18.
C. Gamini Kannangara Ute C. Vothknecht Mats Hansson Diter von Wettstein 《Molecular & general genetics : MGG》1997,254(1):85-92
Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein
preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley
extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid
stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F,
-G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations
from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 × g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored.
Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays
using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution
assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal
RNA, respectively.
Received: 9 September 1996 / Accepted: 22 October 1996 相似文献
19.
New developments in abscisic acid perception and metabolism 总被引:3,自引:0,他引:3
Abscisic acid is a powerful signaling molecule that accumulates in response to abiotic stress. However, no potential receptors that could perceive this increase in abscisic acid had been identified until recent reports of three abscisic acid binding proteins: the nuclear protein Flowering Time Control Locus A, the chloroplast protein Magnesium Protoporphyrin-IX Chelatase H subunit, and the membrane-associated protein G Protein Coupled Receptor 2. Abscisic acid metabolism also has a new and prominent component with the identification of a beta-glucosidase capable of releasing biologically active abscisic acid from inactive abscisic acid-glucose ester in a stress-inducible manner. These observations refocus our attention on the metabolism underlying abscisic acid accumulation, sites of abscisic acid perception, and delivery of abscisic acid to those sites. 相似文献
20.
Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively. 相似文献