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Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Kannangara, C. G.; Vothknecht, U. C.; Hansson, Mats LU and von Wettstein, D. (1997) In Molecular General Genetics 254(1). p.85-92
Abstract
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... (More)
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 x 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. (Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Chlorophyll/biosynthesis, Chloroplasts/metabolism, Genetic Complementation Test, Hordeum/*enzymology, Lyases/*chemistry/metabolism, Rhodobacter sphaeroides/*enzymology, Ribosomes/metabolism
in
Molecular General Genetics
volume
254
issue
1
pages
85 - 92
publisher
Springer
external identifiers
  • scopus:0030903547
ISSN
1432-1874
DOI
10.1007/s004380050394
language
English
LU publication?
no
id
1dd50fcb-f68e-4b4b-97ab-c1796addc57d (old id 8001549)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/9108294
date added to LUP
2015-09-30 14:45:19
date last changed
2017-01-01 04:33:11
@article{1dd50fcb-f68e-4b4b-97ab-c1796addc57d,
  abstract     = {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 x 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.},
  author       = {Kannangara, C. G. and Vothknecht, U. C. and Hansson, Mats and von Wettstein, D.},
  issn         = {1432-1874},
  keyword      = {Chlorophyll/biosynthesis,Chloroplasts/metabolism,Genetic Complementation Test,Hordeum/*enzymology,Lyases/*chemistry/metabolism,Rhodobacter sphaeroides/*enzymology,Ribosomes/metabolism},
  language     = {eng},
  number       = {1},
  pages        = {85--92},
  publisher    = {Springer},
  series       = {Molecular General Genetics},
  title        = {Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD},
  url          = {http://dx.doi.org/10.1007/s004380050394},
  volume       = {254},
  year         = {1997},
}