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ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

Sirijovski, Nick LU ; Olsson, Ulf LU ; Lundqvist, Joakim LU ; Al-Karadaghi, Salam LU ; Willows, Robert D. and Hansson, Mats LU (2006) In Biochemical Journal 400. p.477-484
Abstract
Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA(+) (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase... (More)
Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA(+) (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Rhodobacter, capsulatus, protoporphyrin IX, magnesium chelatase, chlorophyll, ATPases associated with various cellular activities (AAA(+)), bchH
in
Biochemical Journal
volume
400
pages
477 - 484
publisher
Portland Press Limited
external identifiers
  • wos:000242983200011
  • scopus:33845722688
ISSN
0264-6021
DOI
10.1042/BJ20061103
language
English
LU publication?
yes
id
275bda58-3383-4d0f-ac98-b57074a3a9eb (old id 681979)
date added to LUP
2008-01-03 15:56:32
date last changed
2019-02-20 07:43:05
@article{275bda58-3383-4d0f-ac98-b57074a3a9eb,
  abstract     = {Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA(+) (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.},
  author       = {Sirijovski, Nick and Olsson, Ulf and Lundqvist, Joakim and Al-Karadaghi, Salam and Willows, Robert D. and Hansson, Mats},
  issn         = {0264-6021},
  keyword      = {Rhodobacter,capsulatus,protoporphyrin IX,magnesium chelatase,chlorophyll,ATPases associated with various cellular activities (AAA(+)),bchH},
  language     = {eng},
  pages        = {477--484},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact},
  url          = {http://dx.doi.org/10.1042/BJ20061103},
  volume       = {400},
  year         = {2006},
}