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- Filling, C, et al.
(författare)
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Critical residues for structure and catalysis in short-chain dehydrogenases/reductases
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:28, s. 25677-25684
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Tidskriftsartikel (refereegranskat)abstract
- Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Angstrom resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.
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- Filling, C, et al.
(författare)
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Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold
- 2001
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 289:3, s. 712-717
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Tidskriftsartikel (refereegranskat)abstract
- Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3 beta /17 beta -hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-Angstrom resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11 beta -hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.
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- Jornvall, H., et al.
(författare)
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Multiplicity of eukaryotic ADH and other MDR forms
- 2003
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Ingår i: Chemico-Biological Interactions. - 0009-2797 .- 1872-7786. ; 143-144, s. 255-261
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Eukaryotic genomes code for at least eight medium-chain dehydrogenases/reductases (MDR) enzyme families of two types, with and without Zn2+ at the active site. Four families have Zn2+: 'Dimeric alcohol dehydrogenases (ADHs)' (including liver ADHs), 'Tetrameric ADHs' (including the yeast ADHs), 'Cinnamyl ADHs' and 'Polyol DHs'. In the human genome, there are minimally 23 MDR genes, but the list is still growing from further interpretations. Of these, seven genes on chromosome 4 (and three pseudogenes) represent the ADH classes in the gene order IV, I?, Iß, Ia, V, II and III. The lineages leading to human ADH establish five levels of divergence, with nodes at the MDR/short-chain dehydrogenases/reductases (SDR), dimer/tetramer, class III/non-III, further class, and intraclass levels of divergence. These multiplicities allow conclusions on pathways of function for ADHs and suggest this activity to have two roles in addition to its function in metabolism, one of a basic defence nature, the other of regulatory value in higher eukaryotes. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
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