| 1. |
- Hasan, Mahmudul
(författare)
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Structural characterization of proteins to investigate their roles in diseases: Focus on MID & LTA4H
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein molecules are responsible for many biological functions in cells. In order to fulfill their various biological roles, these chain-like molecules must fold into precise three-dimensional shapes. The knowledge of accurate molecular structures is a prerequisite for rational drug design and for structure based functional studies. Getting structural information of proteins can be a very difficult task, especially when it comes to high resolution. Moraxella catarrhalis is widely recognized human-restricted gram-negative bacterium for which it has become clear that it is a true pathogen of both the upper and lower respiratory tract. After Haemophilus influenzae and Streptococcus pneumonia, it is the third most common cause of otitis media in children. The bacterium can directly stimulate B-cells without any recognition of T-cells and it can therefore be classified as a T-cell independent antigen. The mitogenic activity of Moraxella catarhallis is performed by a 2139 residue long outer membrane protein MID. An IgD binding domain (MID962-1200) has been described and the colonization to human respiratory tract cells is mediated by a 150-residue adhesin domain (MID764-913). SAXS studies on the IgD binding domain showed that this domain has an elongated 3-fold organization and that there is the presence of unordered/flexible structures. CD data and prediction of secondary structure for both of the domains indicated the presence of large amounts of (∼33%) ß-sheet and ∼10% α-helix content. Native datasets for MID962-1200 to 2.3 Å resolution and for MID764-913 to 2.7 Å resolution are collected and processed. Vertebrate leukotriene A4 hydrolases are zinc metalloenzymes with an epoxide hydrolase and aminopeptidase activity belonging to the M1 family of aminopeptidases. The human enzyme produces LTB4, a powerful mediator of inflammation and is implicated in a wide variety of rheumatoid diseases. The yeast homolog scLTA4H contains only a rudimentary epoxide hydrolase activity and was shown to undergo a large conformational change upon binding of the inhibitor bestatin. In SAXS studies XlLTA4H shows a more compact form upon bestatin binding, but humLTA4H did not. It was confirmed that the LTA4H from Xenopus is a dimer and that it seems to contract in size upon bestatin binding. In contrast the human enzyme does not show any major difference in SAX scattering patterns upon inhibitor binding and it seems therefore that this enzyme does not display larger conformational changes.
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| 2. |
- Helgstrand, Charlotte, et al.
(författare)
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A Leukotriene A(4) Hydrolase-Related Aminopeptidase from Yeast Undergoes Induced Fit upon Inhibitor Binding.
- 2011
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 406, s. 120-134
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Tidskriftsartikel (refereegranskat)abstract
- Vertebrate leukotriene A(4) hydrolases are bifunctional zinc metalloenzymes with an epoxide hydrolase and an aminopeptidase activity. In contrast, highly homologous enzymes from lower organisms only have the aminopeptidase activity. From sequence comparisons, it is not clear why this difference occurs. In order to obtain more information on the evolutionary relationship between these enzymes and their activities, the structure of a closely related leucine aminopeptidase from Saccharomyces cerevisiae that only shows a very low epoxide hydrolase activity was determined. To investigate the molecular architecture of the active site, the structures of both the native protein and the protein in complex with the aminopeptidase inhibitor bestatin were solved. These structures show a more spacious active site, and the protected cavity in which the labile substrate leukotriene A(4) is bound in the human enzyme is partially obstructed and in other parts is more solvent accessible. Furthermore, the enzyme undergoes induced fit upon binding of the inhibitor bestatin, leading to a movement of the C-terminal domain. The main triggers for the domain movement are a conformational change of Tyr312 and a subtle change in backbone conformation of the PYGAMEN fingerprint region for peptide substrate recognition. This leads to a change in the hydrogen-bonding network pulling the C-terminal domain into a different position. Inasmuch as bestatin is a structural analogue of a leucyl dipeptide and may be regarded as a transition state mimic, our results imply that the enzyme undergoes induced fit during substrate binding and turnover.
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| 3. |
- Stsiapanava, Alena, et al.
(författare)
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Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.
- 2014
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1844:2, s. 439-446
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Tidskriftsartikel (refereegranskat)abstract
- Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.
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