Substrate specificity and inhibitor analyses of human steroid 5β-reductase (AKR1D1).
Human steroid 5β-reductase (aldo-keto reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of Δ(4)-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ(4)-ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5β-steroid metabolites present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the C11 position was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1. By contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.
Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104-6084, USA.
Mo
M
Chen
Jason E
JE
Drury
Trevor M
TM
Penning
D001647:Bile Acids and Salts / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007664:Ketosteroids / Q000378:metabolism; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D007664:Ketosteroids; D010088:Oxidoreductases; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase
J Biol Chem. 2008 Jun 13;283(24):16830-9
18407998
Biochemistry. 1998 Mar 10;37(10):3538-48
9521675
Biochem Pharmacol. 2008 Jan 15;75(2):484-93
17950253
Biochemistry. 2004 Sep 28;43(38):12028-37
15379543
Biochemistry. 1992 May 26;31(20):4737-49
1591235
Mol Pharmacol. 2005 Jan;67(1):60-8
15475569
Mol Hum Reprod. 2005 Jul;11(7):495-501
16123077
Biochemistry. 1995 Nov 7;34(44):14356-65
7578039
Biochemistry. 2008 Dec 23;47(51):13537-46
19075558
Phytochemistry. 2007 Mar;68(6):853-64
17184799
Steroids. 1994 Jun;59(6):352-6
7940612
Cancer Res. 2004 Mar 1;64(5):1802-10
14996743
Proc Natl Acad Sci U S A. 1967 May;57(5):1463-7
5231752
Steroids. 2008 Apr;73(4):417-23
18243262
Biochem J. 1997 Sep 15;326 ( Pt 3):625-36
9307009
Biochemistry. 1998 Jul 7;37(27):9695-703
9657682
Biochim Biophys Acta. 1969;185(2):269-86
4980133
Biochim Biophys Acta. 2001 Jan 26;1517(2):228-35
11342103
Steroids. 1990 Nov;55(11):495-500
2075615
J Biol Chem. 2000 May 19;275(20):15122-7
10748001
Arch Biochem Biophys. 2007 Aug 15;464(2):241-50
17537398
Biochemistry. 2006 Oct 31;45(43):13054-63
17059222
J Biol Chem. 1996 Nov 22;271(47):30190-8
8939970
Clin Res. 1987 Jan;35(1):1-12
2434280
Eur J Biochem. 1994 Jan 15;219(1-2):357-63
7508385
J Biol Chem. 2009 Jul 24;284(30):19786-90
19515843
Structure. 1997 Jun 15;5(6):799-812
9261071
Proc Natl Acad Sci U S A. 1967 Sep;58(3):985-90
5233854
J Biol Chem. 2004 May 28;279(22):22908-12
15044432
J Hepatol. 2010 May;52(5):705-11
20347173
Eur J Biochem. 1967 Nov;2(4):493-502
4384270
J Biol Chem. 2007 Nov 16;282(46):33484-33493
17848571
Science. 1992 Jul 3;257(5066):81-4
1621098
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12208-13
9770465
Biochemistry. 2008 Aug 12;47(32):8261-70
18624455