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- Willows, R D, et al.
(författare)
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EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer
- 2004
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1095-8657 .- 1047-8477. ; 146:1-2, s. 227-233
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Tidskriftsartikel (refereegranskat)abstract
- BchI, belonging to the AAA+-protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA+-protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA+-proteins are Mg2+-dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI–ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at ~30 Å and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30 Å.
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| 2. |
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| 3. |
- Fodje, Michel, et al.
(författare)
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Interplay Between an AAA Module and an Integrin I Domain May Regulate the Function of Magnesium Chelatase
- 2001
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 311:1, s. 111-122
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Tidskriftsartikel (refereegranskat)abstract
- In chlorophyll biosynthesis, insertion of Mg2+ into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (Rfree = 24.5 %). It belongs to the chaperone-like ''ATPase associated with a variety of cellular activities'' (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.
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| 4. |
- Hansson, Andreas, et al.
(författare)
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Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers
- 2002
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 99:21, s. 13944-13949
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Tidskriftsartikel (refereegranskat)abstract
- Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA+) family of proteins. AAA+ proteins are Mg2+-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.
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| 6. |
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| 7. |
- Willows, R D, et al.
(författare)
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Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit
- 1999
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D55, s. 689-690
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Tidskriftsartikel (refereegranskat)abstract
- The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 Å. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 Å.
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