The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition

Abstract

7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP + by X-ray diffraction, which, as a tetramer, possesses the typical α/β folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axisrelated subunits. Significantly, we captured an active state of the NADP + , confirming that nicotinamide moiety of NADP + act as electron carrier in the dehydrogenation. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, furthermore, we find that the guanidinium of Arg38 can form the stable cation-π interaction with the adenine ring of NADP + , and the cation-π interaction and hydrogen bonds between Arg38 and NADP + have a significant anchor effect on the cofactor binding to CA 7α-HSDH.

The effects of macromolecular crowding and surface charge on the properties of an immobilized enzyme: activity, thermal stability, catalytic efficiency and reusability

“…The expression and purication of 7a-HSDH (EC 1.1.1.159) and 7b-HSDH (EC 1.1.1.201) were performed using a GST gene fusion system. 26 TCDCA and TUDCA standard samples were purchased from the National Institutes for Food and Drug Control (Beijing, China). Sodium taurine-7ketolithocholic acid (T-7-KLCA) was synthesized by our laboratory.…”

“…The electrostatic calculations for the surface of 7a-HSDH and 7b-HSDH indicate that the surface is negatively charged at pH 7, which is Size exclusion chromatography (SEC) was used to determine the molecular weight distribution and the results indicate that in the CA, 7a-HSDH mainly exists as a dimer. 26 Therefore, the widest dimensions of 7a-HSDH and 7b-HSDH were calculated by the farthest distance between two amino acids with 7a-HSDH and 7b-HSDH existing as dimers (Fig. 1e and f).…”

Molecular Mechanism Study on Stereo-Selectivity of α or β Hydroxysteroid Dehydrogenases

“…However, the dihedral angle PA-O3-PN-O5D (-80.6 • ) in the 7β-HSDH binary is apparently different compared with others, which may lead to the difference of substrate binding pocket. Another crystal structure of 7α-HSDH from Clostridium absonum was obtained as a tetramer in the study by Lou et al, which exhibited a coenzyme-dependent of NADP(H) [94]. The dihedral angle PA-O3-PN-O5D in the pyrophosphate region is −143.4 • (Figure 8), which is similar to 7α-HSDH-NAD + complex (-147.8 • ) from E. coli, although the coenzymes of these two crystals are completely different.…”

“…In the nicotinamide ring binding region, the interactions between nicotinamide ribose and residues seem to be conserved. Motifs Tyr-X-X-X-Lys [89][90][91][92][93][94] and Thr/Ile-X-Thr have conserved interactions with NAD(P)(H) (Figure 7 and Table 2). The hydrogen bonds existed among PN-phosphate group of pyrophosphate region, nicotinamide, and Thr contribute to maintaining the orientation of pro-S side of nicotinamide towards catalytic sites, for example, the interactions…”

“…Other amino acid residues participating in the catalytic reactions were proposed. Lou et al elaborated the mechanism in the study of 7α-HSDH from Clostridium absonum [94], it was found that the serine in Ser-Tyr-Lys triad was replaced by threonine, without a complete activity loss of 7α-HSDH. In the study of 3(20)β-HSDH, it was found that the Asp participated in the catalysis, which formed an active site tetrad Ser-Tyr-Lys-Asp.…”