Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4).
DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3alpha-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3beta-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.
Endo
Satoshi
S
Laboratory of Biochemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan. sendo@gifu-pu.ac.jp
Maeda
Satoshi
S
Matsunaga
Toshiyuki
T
Dhagat
Urmi
U
El-Kabbani
Ossama
O
Tanaka
Nobutada
N
Nakamura
Kazuo T
KT
Tajima
Kazuo
K
Hara
Akira
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D004285:Dogs; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism*; D016415:Sequence Alignment; D013552:Swine; D014162:Transfection
D011994:Recombinant Proteins; D006884:Hydroxybutyrate Dehydrogenase; C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D064429:Fatty Acid Synthases; D012172:Retinaldehyde