Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold.
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 3beta/17beta-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-A 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 11beta-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.
Filling
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Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Nordling
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Benach
J
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Berndt
K D
KD
Ladenstein
R
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Jörnvall
H
H
Oppermann
U
U
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000502:physiology*; D002942:Circular Dichroism; D017124:Conserved Sequence; D006031:Glycosylation; D019791:Guanidine / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D001216:Asparagine; D010088:Oxidoreductases; D015067:17-Hydroxysteroid Dehydrogenases; D006913:Hydroxysteroid Dehydrogenases; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D019791:Guanidine