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- Hosseini Maaf, Bahram, et al.
(författare)
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Structural basis for red cell phenotypic changes in newly identified, naturally occurring subgroup mutants of the human blood group B glycosyltransferase.
- 2007
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Ingår i: Transfusion. - : Wiley. - 1537-2995 .- 0041-1132. ; 47:5, s. 864-875
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Four amino-acid-changing polymorphisms differentiate the blood group A and B alleles. Multiple missense mutations are associated with weak expression of A and B antigens but the structural changes causing subgroups have not been studied. STUDY DESIGN AND METHODS: Individuals or families having serologically weak B antigen on their red cells were studied. Alleles were characterized by sequencing of exons 1 through 7 in the ABO gene. Single crystal X-ray diffraction, three-dimensional-structure molecular modeling, and enzyme kinetics showed the effects of the B allele mutations on the glycosyltransferases. RESULTS: Seven unrelated individuals with weak B phenotypes possessed seven different B alleles, five of which are new and result in substitution of highly conserved amino acids: M189V, I192T, F216I, D262N, and A268T. One of these (F216I) was due to a hybrid allele resulting from recombination between B and O-1v alleles. The two other alleles were recently described in other ethnic groups and result in V175M and L232P. The first crystal-structure determination (A268T) of a subgroup glycosyltransferase and molecular modeling (F216I, D262N, L232P) indicated conformational changes in the enzyme that could explain the diminished enzyme activity. The effect of three mutations could not be visualized since they occur in a disordered loop. CONCLUSION: The genetic background for B-w phenotypes is very heterogeneous but usually arises through seemingly random missense mutations throughout the last ABO exon. The targeted amino acid residues, however, are well conserved during evolution. Based on analysis of the resulting structural changes in the glycosyltransferase, the mutations are likely to disrupt molecular bonds of importance for enzymatic function.
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| 2. |
- Moe, Agnes, 1990-
(författare)
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Role of respiratory supercomplexes : Electronic connection between complexes III and IV
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the final step of cellular respiration, electrons are transferred through the respiratory chain to reduce molecular oxygen to water. The energy released in this chain is used to maintain a proton electrochemical gradient across the cell membrane, which is used, for example, by the ATP synthase to produce ATP. The enzyme complexes of the respiratory chain are known to organize in supramolecular assemblies, so-called respiratory supercomplexes.In this work we investigated the functional significance of respiratory supercomplexes consisting of complexes III and IV in mitochondria. By combining structural and kinetic studies we showed that at the commonly assumed "physiological" ionic strength of 150 mM monovalent salt, the water-soluble cyt. c associates with the negatively charged surface of III2-IV1-2 supercomplexes in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. The data showed that one cyt. c diffuses in 2D, between complexes III and IV, indicating a kinetic advantage of forming supercomplexes. These studies also showed different relative orientation of the individual complexes in the supercomplexes from the two yeast species, indicating that 2D diffusion is a general mechanism, not limited to a specific relative orientation of complexes III and IV. More recent data in the literature indicate that a more realistic mimic of intracellular conditions is a monovalent salt concentration of 20 mM. We showed that under these conditions two cyt. c molecules bind simultaneously to the supercomplex. This result further supports a kinetic advantage of forming supercomplexes.We also determined the cryo-EM structure of the obligate III2-IV2 supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure revealed an electronic connection between complexes III and IV by a di-heme cyt. cc subunit. The structure also showed that complexes III and IV are structurally intertwined and strongly connected with unique features conserved in the phylum actinobacteria.
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| 3. |
- Persson, Mattias, et al.
(författare)
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Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:13, s. 9564-9570
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Tidskriftsartikel (refereegranskat)abstract
- Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fuc alpha(1-2)Gal beta-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic (DVD213)-D-211 motif that coordinates to a Mn2+ ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the (211)DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B,1 variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn2+. Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity.
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