Structure-Function Studies of FR-1

Abstract

The aldo-keto reductase (AKR) gene superfamily represents a collection of proteins expressed in a wide variety of plants, animals, yeast, and procaryotic organisms. Most AKRs were originally identified as enzymes capable of catalyzing the NADPH-dependent reduction of carbonyl groups contained in a broad range of substrates (Bachur, 1976). However, recent genetic studies mediated by genome and expression sequencing approaches have identified several new members of the AKR superfamily. Many of these new proteins are characterized by high sequence homology to AKR enzymes although little or no information is available about their potential catalytic activities. One such new protein, designated FR-1* was identified as the product of a gene upregulated in serum-starved mouse fibroblasts following treatment with fibroblast growth factor I (FGF-I) (Donohue et al., 1994). High amino acid sequence identity (~70%) was observed between FR-1 and aldose reductase as well as other AKRs. Many amino acid residues known to contribute to the catalytic mechanism in other AKR enzymes including aldose reductase (AKRlB I), aldehyde reductase (AKRIAI) and 3a.-hydroxysteroid dehydrogenase (AKR1C9) are conserved in FR-l. These residues include Tyr-48, His-ll 0, Lys-77 and Asp-43 (numbering is that of aldose reductase) (Barski et al. , 1995; Pawlowski & Penning, 1994; Schlegel et al., 1998; Tarle et al. , 1993). The present study was undertaken to evaluate whether FR -1 is a catalyst of carbonyl reduction and to measure the affinity of FR-1 for various ligands such as nucleotide cofactors, carbonyl substrates and aldose reductase inhibitors. Our studies show that FR-1 catalyzes the NADPH-dependent reduction of substrates representative of diverse structural classes of aliphatic and aromatic aldehydes. Both saturated and unsaturated aldehydes were excellent substrates. Unlike aldose reductase and aldehyde reductase, FR-1 catalyzed the reduction of simple ketones such as acetone and butanone; however virtually no catalytic activity could be detected using steroid and aldose substrates. FR-1 was inhibited by various aldose reductase inhibitors in a manner similar to human aldose reductase. Besides being an excellent substrate, 4-hydroxy-2-nonenal (HNE) inactivated the enzyme through a mechanism involving Michael addition to Cys-298.