Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

Abstract

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3a,20/3-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the a/( fold characteristic ofthe dinucleotide binding region. The fold of the rest of the subunit, the quarternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3a-and 2013-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. (1), which includes 11f,-hydroxysteroid (llP-HSD) (2), 7a-hydroxysteroid (3), and 15-hydroxyprostaglandin dehydrogenases (4) from mammals; glucose (5) and ribitol (6) dehydrogenases, as well as a putative nodulation factor (7) from bacteria; and an ADH (8) from insects. Enzymes belonging to this family have -250 amino acid residues, similar coenzyme specificity, and partial sequence homology. Although more than 40 crystal structures of =15 types of NAD(H)-and NADP(H)-linked dehydrogenase enzymes have been determined at medium-to-high resolution (9), to our knowledge no x-ray crystallographic study describing the three-dimensional structure of a dehydrogenase belonging to this short-chain class has been reported. This is only the third structure of an enzyme for which steroids are the substrate to be determined by x-ray diffraction techniques. A lowresolution structure of keto-steroid isomerase (10) and the refined structure of cholesterol oxidase (11) have been published.To account for the ability of 3a,20f3-HSD to transfer a hydride to either end of a steroid molecule, "one steroid-two cofactor sites" and "two steroid orientations-one cofactor site" models (12) have been proposed. When analyzed in conjunction with sequence homology studies, the threedimensional structured especially at the cofactor binding and the substrate binding regions, offers further insight concerning the significance of conserved residues and their possible roles in substrate specificity and overall enzyme function. MATERIALS AND METHODSThe crystals, grown in the presence of 4 mM NADH, belong to the space group P43212 having unit cell dimensions a = 106.2 A and c = 203.8 A and contain one full tetramer (106 kDa) in the asymmetric unit (13 .091], was collected on film from six crystals at the Cornell High Energy Synchrotron Source and processed by using Rossmann's program at Purdue University. The area detector data to 3 A and film data between 3 and 2.6 A were merged into a composite native data set. The Hg derivatives each had a single major binding site per subunit, whereas the Au reagent gave a "multip...

Effect of site‐directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase

“…Following the three-dimensional structure of the homologous region in the 3a,20/3-hydroxysteroid dehydrogenase, these two glycines are clearly positioned on the outer surface of the molecule, at the hinge of an a-j? connecting segment [5]. According to these data, both could be crucial for the spatial configuration of the enzyme.…”

“…Lys15'j is invariably present in ADH of 36 species of Drosophila and also in all analyzed members of the short-chain family. The high degree of conservation, the proximity to the catalytic Tyr and the chemical features of its side chain clearly suggested that this Lys could well be enclosed in the catalytic cleft [5], providing an alkaline environment for catalysis. Its E-NH, group, if suitably located with respect to Tyr, could have the effect of lowering the apparent pK, of the phenolic group, thus facilitating the hydride transfer reaction at much lower, physiological pH.…”

Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase

“…Typically, residue identities between the different enzymes are 20±30%, indicating early deviation from a common origin. Nevertheless, crystallographic analysis of so far two bacterial [9,13] and three mammalian enzymes [10±12] has revealed the same Rossmann fold secondary structure and a highly conserved three-dimensional architecture. By combining primary and tertiary structural information it has been possible to identify several sequence motifs that are conserved in most SDRs.…”

“…The functional role of the reactive cysteine, however, remains speculative. Based on the known tertiary structure of the bacterial 3a/20b-hydroxysteroid dehydrogenase [9], Jo Èrnvall and colleagues have constructed a three-dimensional model of human carbonyl reductase [25]. Although the model has some uncertainties, particularly in the C-terminal part of the peptide chain, it clearly identifies the residues constituting the a-helices and b-pleated sheets of the Rossmann fold and positions Cys227 in the C-terminal part of the bF strand, where it precedes a highly conserved proline residue.…”

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

“…The N-terminus of the molecule is shown in blue, and the C-terminus in yellow. The nomenclature for secondary structural elements was adapted from Ghosh et al 17 The missing region is depicted by a dashed magenta line. bind in an extended conformation to residues forming the base of the active cleft between the two domains.…”