Characterization of rat and mouse NAD+-dependent 3α/17β/20α-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis
Section snippets
Chemicals
Steroids were obtained from Steraloids (Newport, RI) and Sigma Chemicals (Ann Arbor, MI); prostaglandins were from Cayman Chemicals (Ann Arbor, MI, U.S.A.); resins for column chromatography were from Amersham Biosciences (Piscataway, NJ); and a pCR T7/CT-TOPO TA expression kit and Escherichia coli BL21 (DE3) pLysS were from Invitrogen (Carlsbad, CA). Pfu DNA polymerase and Taq DNA polymerase were purchased from Stratagene (La Jolla, CA) and Takara (Kusatsu, Japan), respectively. 6-tert
Purification and properties of recombinant AKR1C16 and AKR1C13
The amino acid sequences deduced from the isolated cDNAs for AKR1C16 and AKR1C13 differed by a few amino acids from those of the previously reported cDNAs for the enzymes [12], [13]. However, they are identical to those of the proteins encoded in their rat and mouse genes (Accession No. RGD: 1308232 and ENSMUSG00000021213, respectively). The extracts of the E. coli cells transfected with the expression plasmids harboring the cDNAs for the two enzymes over-expressed 37-kDa proteins, which were
Discussion
The enzymatic properties of recombinant AKR1C16 and AKR1C13 characterized in the present study indicate that the two proteins are NAD+-preferring enzymes with broad substrate specificity for 20α-, 17β- and 3α-hydroxysteroids, and non-steroidal alcohols. Although the kcat values for most hydroxysteroid substrates were less than 1 min−1, the kcat/Km values for some hydroxysteroids are comparable to those for the good non-steroidal alcohol substrates. The two enzymes may physiologically act as
Acknowledgments
We thank Ms. Urmi Dhagat for her assistance in preparing the figure for the modeled structures of the AKRs (1C16, 1C17 and 1C24). This work was supported by a Grant-in-Aid for Young Scientists (Start-up) from the Japan Society for the Promotion of Science.
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Cited by (10)
Characterization of aldo-keto reductase 1C subfamily members encoded in two rat genes (akr1c19 and RGD1564865). Relationship to 9-hydroxyprostaglandin dehydrogenase
2021, Archives of Biochemistry and BiophysicsCitation Excerpt :By contrast, most of the R1C19 substrates were not oxidized by 20HSDL, which instead exhibited dehydrogenase activities towards trans-benzene dihydrodiol and (1S,2S)-trans-1,2-cyclohexanediol. Neither R1C19 nor 20HSDL oxidized other xenobiotic alcohols and hydroxysteroids (listed in Table 2), most of which are substrates of other rat AKR1Cs [13,15–20]. It should be noted that 20HSDL does not accept 20α-hydroxysteroids as its substrates, contrary to the current protein name encoded in the RGD1564865 gene.
Mouse Akr1cl gene product is a prostaglandin D<inf>2</inf> 11-ketoreductase with strict substrate specificity
2019, Archives of Biochemistry and BiophysicsIdentification of a determinant for strict NADP(H)-specificity and high sensitivity to mixed-type steroid inhibitor of rabbit aldo-keto reductase 1C33 by site-directed mutagenesis
2015, Archives of Biochemistry and BiophysicsCitation Excerpt :Previous studies have shown that salt bridges between K270 and/or R276 and the NADPH 2′-phosphate are critical for the preference for NADP(H) [1,4]. The opposite preference for NAD(H) to NADP(H) is observed in several AKRs, in which the K270 and/or R276 in the NADP(H)-preferring AKRs are replaced with Q and E, respectively [11–15]. The key roles of Q270 and E276 in the NAD(H) preference are demonstrated by site-directed mutagenesis of the residues in rat NAD+-dependent 3α-hydroxysteroid dehydrogenase (HSD) [13].
Characterization of rabbit morphine 6-dehydrogenase and two NAD <sup>+</sup>-dependent 3α(17β)-hydroxysteroid dehydrogenases
2013, Archives of Biochemistry and BiophysicsCitation Excerpt :The purified preparations of the recombinant AKR1C26, AKR1C27 and AKR1C28 showed single 37-kDa protein bands on the SDS–PAGE analysis, in which no other protein band was observed. The enzymes exhibited NAD+-linked dehydrogenase activity towards S-tetralol that is an excellent substrate for the above HSDs from other species [14–16]. The specific activities of AKR1C26, AKR1C27 and AKR1C28 were 0.033, 0.443 and 0.127 unit/mg, respectively.
Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8. A hyperthermostable aldose-1-dehydrogenase with broad substrate specificity
2009, Chemico-Biological InteractionsCitation Excerpt :TAD possesses a unique ability to oxidize non-sugar alicyclic alcohols. This ability has not been reported for bacterial dehydrogenases for aldoses, although it is observed for mammalian NADPH-dependent lung carbonyl reductases [30,31] in the SDR superfamily and NAD(P)+-dependent hydroxysteroid dehydrogenases (HSDs) [32,33] belonging to the AKR superfamily. Dehydrogenases for the C1-hydroxyl groups of d-glucose [25,34], d-galactose [35], l-arabinose [27] and d-fucose [36] are involved in the non-phosphorylative Enter-Doudoroff pathway or its alternative metabolic pathways in archaea and several bacteria.
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Present address: Hospital for Sick Children, Toronto, Ont., Canada M5G 1X8.