| 1. |
- Dufe, Veronica T, et al.
(författare)
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A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 1-amino-oxy-3-aminopropane.
- 2007
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Ingår i: Biochem J. - 1470-8728. ; 405:2, s. 261-268
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Tidskriftsartikel (refereegranskat)abstract
- The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a K(i) value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5'-phosphate and the irreversible inhibitor alpha-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5'-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC-APA-PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158-168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.
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| 2. |
- Sirijovski, Nick, et al.
(författare)
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ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact
- 2006
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Ingår i: Biochemical Journal. - 0264-6021. ; 400, s. 477-484
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Tidskriftsartikel (refereegranskat)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.
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| 3. |
- Tamu Dufe, Veronica, et al.
(författare)
-
A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 3-aminooxy-1-aminopropane
- 2007
-
Ingår i: Biochemical Journal. - 0264-6021. ; 405:Pt 2, s. 261-268
-
Tidskriftsartikel (refereegranskat)abstract
- The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a Ki value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5′-phosphate and the irreversible inhibitor α-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5′-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC–APA–PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158–168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.
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