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J. Biol. Chem., Vol. 278, Issue 41, 40079-40087, October 10, 2003

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Identification of a Mouse Short-chain Dehydrogenase/Reductase Gene, Retinol Dehydrogenase-similar

FUNCTION OF NON-CATALYTIC AMINO ACID RESIDUES IN ENZYME ACTIVITY^*

Min-Sun Song, Weiguo Chen , Min Zhang and Joseph L. Napoli 

From the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720

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 3-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-S3 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 G²⁰⁸FKTCVTSSD for RDH-S sequence F²⁰⁸-FLTGMASSA. RDH-S3 catalyzed all-trans-retinol and 5-androstane-3,17-diol (3-adiol) metabolism 60–70% as efficiently (V_m/K_m) as RDH1. Conversely, substituting RDH-S sequence F²⁰⁸FLTGMASSA into RDH1 produced a chimera (viz. C3) that was inactive with all-trans-retinol, but was 4-fold more efficient with 3-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 2.4-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 3-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.

Received for publication, May 9, 2003 , and in revised form, July 10, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY046408.

^* This work was supported by National Institutes of Health Grant DK36870. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Div. of Allergy and Immunology, Dept. of Pediatrics, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.

To whom correspondence should be addressed: Dept. of Nutritional Sciences and Toxicology, University of California, 119 Morgan Hall, MC3104, Berkeley, CA 94720. Tel.: 510-642-0809; Fax: 510-642-0535; E-mail: jna{at}uclink4.berkeley.edu.

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