Crystal structure of an apo 7α-hydroxysteroid dehydrogenase reveals key structural changes induced by substrate and co-factor binding. Issue 212 (September 2021)

• Record Type: Journal Article
• Authors: Kim, Ki-Hwa; Lee, Chang Woo; Pardhe, Bashu Dev; Hwang, Jisub; Do, Hackwon; Lee, Yung Mi; Lee, Jun Hyuck; Oh, Tae-Jin
• Is Part Of: Journal of steroid biochemistry and molecular biology. Issue 212(2021)
• Publication Date: 2021-09
• Subjects: 7-KLCA 7-ketolithocholic acid -- ADH alcohol dehydrogenase -- Bme-7α-HSDH Brucella melitensis-7α-hydroxysteroid dehydrogenase -- Cab-7α-HSDH Clostridium absonum 7α-hydroxysteroid dehydrogenase -- CDCA chenodeoxycholic acid -- Eco-7α-HSDH Escherichia coli 7α-hydroxysteroid dehydrogenase -- Eco-ADH Escherichia coli alcohol dehydrogenase -- LC liquid chromatography -- MS mass spectrometry -- PDB Protein Data Bank -- UDCA ursodeoxycholic acid; Crystal structure -- Conformational change -- 7α-hydroxysteroid dehydrogenase -- X-ray crystallography; Steroid hormones -- Periodicals; Biochemistry -- Periodicals; Hormones -- Periodicals; Molecular Biology -- Periodicals; Hormones stéroïdes -- Périodiques; Steroid hormones; Periodicals; 572.579
• DOI: 10.1016/j.jsbmb.2021.105945
• Languages: English
• ISSNs: 0960-0760

Abstract

Graphical abstract: Highlights: The apo 7α-HSDH crystal structure was determined at 2.7 Å resolution. The open structure is similar to that of the binary-complex structure (PDB: 1AHH). Eco -ADH was employed for NAD + regeneration to promote Eco -7α-HSDH activity. Abstract: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) catalyzes the dehydrogenation of a hydroxyl group at the 7α position in steroid substrates using NAD + or NADP + as a co-factor. Although studies have determined the binary and ternary complex structures, detailed structural changes induced by ligand and co-factor binding remain unclear, because ligand-free structures are not yet available. Here, we present the crystal structure of apo 7α-HSDH from Escherichia coli ( Eco -7α-HSDH) at 2.7 Å resolution. We found that the apo form undergoes substantial conformational changes in the β4-α4 loop, α7-α8 helices, and C-terminus loop among the four subunits comprising the tetramer. Furthermore, a comparison of the apo structure with the binary (NAD + )-complex and ternary (NADH and 7-oxoglycochenodeoxycholic acid)-complex Eco -7α-HSDH structures revealed that only the ternary-complex structure has a fully closed conformation, whereas the binary-complex and apo structures have a semi-closed or open conformation. This open-to-closed transition forces several catalytically important residues (S146, Y159, and K163) into correct positions for catalysis. To confirm the catalytic activity, we used alcohol dehydrogenase for NAD + regeneration to allow efficient conversion of chenodeoxycholic acid to 7-ketolithocholic acid by Eco -7α-HSDH. These findings demonstrate that apo Eco -7α-HSDH exhibits intrinsically flexible characteristics with an open conformation. This structural information provides novel insight into the 7α-HSDH reaction mechanism.