| 1. |
- 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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| 2. |
- Fodje, Michel, et al.
(författare)
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Occurrence, conformational features and amino acid propensities for the pi-helix.
- 2002
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Ingår i: Protein Engineering. - 1460-213X. ; 15:5, s. 353-358
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Tidskriftsartikel (refereegranskat)abstract
- The most abundant helix type in proteins is the alpha-helix, accounting for about 31% of amino acid secondary structure states, while the 3(10)-helix accounts for about 4%. The pi-helix appears to be extremely rare and is considered to be unstable. Existing secondary structure definition methods find very few within the Protein Data Bank. Using an improved pi-helix definition algorithm to search a non-redundant subset of high-resolution and well-refined protein structures, we found that almost every tenth protein contained a pi-helix. This enabled us to show for the first time that the pi-helix has structural parameters that are different from the hypothesized model values. It also has distinctive amino acid preferences and it is conserved within functionally related proteins. Features that may contribute to the stability of the pi-helical structure have also been identified. In addition to hydrogen bonds, several other factors contribute to the stability of pi-helices. The pi-helix may have some functional advantages over other helical structures. Thus, we describe cases where the side chains of functionally important residues at every fourth position within a pi-helix could be aligned and brought close together in a way that would not be allowed by any other helix type.
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| 3. |
- Fodje, Michel
(författare)
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Structure and mechanism of iron and magnesium chelatases - at the heme-chlorophyll branch-point
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tetrapyrroles are synthesised through a single branched biosynthetic pathway. Heme and chlorophyll are synthesised from the common intermediate protoporphyrin IX at the heme-chlorophyll branch-point. Ferrochelatase catalyses the synthesis of heme by inserting Fe(II) into the protoporphyrin macro-cycle while magnesium chelatase, a three-subunit enzyme, catalyses the first unique step in chlorophyll biosynthesis by inserting Mg(II). The structure of ferrochelatase has been determined both in apo form (1.8 Å) and in complex with the inhibitor N-methyl-mesoporphyrin (1.9 Å). The structure is made up of two Rossmann type domains with an active-site cleft between them. The protein induces a distortion in the porphyrin macro-cycle on binding, exposing the pyrrolenine nitrogen atoms to the in-coming metal. Metal binding studies with substrate (Zn) and inhibitor (Cd) ions reveal the metal binding residues as being the His-Glu couple, H183 and E264. Substrate and inhibitor metal ions showed different coordination geometries at the His-Glu couple and the inhibitor was bound at an additional site. A fully hydrated Mg(II) ion is bound to a pi-helix close to the active-site cleft and interacts with the metal bound at the His-Glu couple. An improved pi-helix definition algorithm enabled detailed analysis of the occurrence, conformational features, amino acid propensities and functional relevance of the pi-helix in proteins structures. The pi-helix was found to be more abundant than previously believed, and possessed unique sequence and structural features that are functionally important, as exemplified by the Mg(II) binding site of ferrochelatase. The structure of the ATPase subunit of magnesium chelatase has been determined to 2.1 Å resolution. It belongs to the AAA+ family but possesses a unique arrangement of domains. Electron micrographs of solutions of this subunit in the presence of ATP revealed ring-like structures akin to AAA hexamers. A model is proposed for the initial magnesium chelatase complex, which involves a two-tiered hexameric ring.
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| 4. |
- Lecerof, David, et al.
(författare)
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Metal binding to Bacillus subtilis ferrochelatase and interaction between metal sites.
- 2003
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 1432-1327 .- 0949-8257. ; 8:4, s. 452-458
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Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn2+, which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd2+, an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg2+, which has been shown to bind close to the surface at 7 Å from His183, was largely absent from its site. Activity measurements demonstrate that Mg2+ has a stimulatory effect on the enzyme, lowering KM for Zn2+ from 55 to 24 µM. Changing one of the Mg2+ binding residues, Glu272, to serine abolishes the effect of Mg2+. It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg2+ site may stimulate metal release from the protein ligands and its insertion into the porphyrin.
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| 5. |
- Lecerof, David, et al.
(författare)
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Structural and Mechanistic Basis of Porphyrin Metallation by Ferrochelatase
- 2000
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 297:1, s. 221-232
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Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase, the enzyme catalyzing metallation of protoporphyrin IX at the terminal step of heme biosynthesis, was co-crystallized with an isomer mixture of the potent inhibitor N-methylmesoporphyrin (N-MeMP). The X-ray structure revealed the active site of the enzyme, to which only one of the isomers was bound, and for the first time allowed characterization of the mode of porphyrin macrocycle distortion by ferrochelatase. Crystallization of ferrochelatase and N-MeMP in the presence of Cu2+ leads to metallation and demethylation of N-MeMP. A mechanism of porphyrin distortion is proposed, which assumes that the enzyme holds pyrrole rings B, C and D in a vice-like grip and forces a 36 o tilt on ring A.
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| 6. |
- Shipovskov, Stepan, et al.
(författare)
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Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: Implications for the catalytic reaction mechanism
- 2005
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 352:5, s. 1081-1090
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Tidskriftsartikel (refereegranskat)abstract
- Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferrochelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to noncatalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the Km values for Zn(II), (17 mu M) and Cu(II) (170 mu M) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.(c) 2005 Elsevier Ltd. All rights reserved.
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