Identification of a mouse short-chain dehydrogenase/reductase gene, retinol dehydrogenase-similar - Function of non-catalytic amino acid residues in enzyme activity

We report a mouse short-chain dehydrogenase/reductase (SDR), retinol dehydrogenase-similar (RDH-S), with intense mRNA expression in liver and kidney. The RDH-S gene localizes to chromosome 10D3 with the SDR subfamily that catalyzes metabolism of retinoids and 3alpha-hydroxysteroids. RDH-S has no activity with prototypical retinoid/steroid substrates, despite 92% amino acid similarity to mouse RDH1. This afforded the opportunity to analyze for functions of non-catalytic SDR residues. We produced RDH-SDelta3 by mutating RDH-S to remove an "additional" Asn residue relative to RDH1 in its center, to convert three residues into RDH1 residues (L121P, S122N, and Q123E), and to substitute RDH1 sequence G(208)FKTCVTSSD for RDH-S sequence F(208)FLTGMASSA. RDH-SDelta3 catalyzed all-trans-retinol and 5alpha-androstane-3alpha,17alpha-diol (3alpha-adiol) metabolism 60-70% as efficiently (V-m/K-m) as RDH1. Conversely, substituting RDH-S sequence F(208)FLTGMASSA into RDH1 produced a chimera ( viz. C3) that was inactive with all-trans-retinol, but was 4-fold more efficient with 3alpha-adiol. A single RDH1 mutation in the C3 region (K210L) reduced efficiency for all-trans-retinol by > 1250-fold. In contrast, the C3 area mutation C212G enhanced efficiency with all-trans-retinol by similar to2.4-fold. This represents a > 6000-fold difference in catalytic efficiency for two enzymes that differ by a single non-catalytic amino acid residue. Another chimera (viz. C5) retained efficiency with all-trans-retinol, but was not saturated and was weakly active with 3alpha-adiol, stemming from three residue differences (K224Q, K229Q, and A230T). The residues studied contribute to the substrate-binding pocket: molecular modeling indicated that they would affect orientation of substrates with the catalytic residues. These data report a new member of the SDR gene family, provide insight into the function of non-catalytic SDR residues, and illustrate that limited changes in the multifunctional SDR yield major alterations in substrate specificity and/or catalytic efficiency.