Abstract
3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD+ causes the appearance of T188-P191 in the binary complex. This study determines the functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis. A stopped-flow study reveals that the rate-limiting step in the reaction is the release of the NADH. The mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in kcat, Kandrosterone and KiNAD and equal primary isotope effects of DV and D(V/K). Therefore, these mutants increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD+ binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis.
Highlights
P185A, P185G, T188A and T188S 3a-HSD/CRs were assessed by circular dichroism (CD) spectroscopy by measuring the ellipticity in the 190– 250 nm range at room temperature
No difference in the CD spectra was observed for the T188 mutants, but an increase in the intensity at 222 nm was observed for P185 mutants, compared to that of the wild-type enzyme, which indicates an increase in the ahelix (Figure 3)
The addition of 100 mM NADH to the P185 and T188 mutated enzymes to form E-NADH binary complex and the further addition of 50 mM androsterone to form E-NADHandrosterone ternary complex has a CD spectrum similar to that of the apoenzyme, which suggests that there is no further change in the secondary structure of mutants induced by the ligands
Summary
3a-Hydroxysteroid dehydrogenase/carbonyl reductase (3aHSD/CR; EC1.1.1.50) from Comamonas testosteroni, reversibly catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. In many SDR enzymes, the substrate-binding loop is involved in the recognition of substrate structure and demonstrates an intrinsic flexibility It is almost completely disordered in the absence of a bound cofactor and substrate, but becomes ordered after substrate binding [17,18,19,20,21]. The motion of the loop is involved in binding with the ligands, which facilitates a reaction by an optimal interaction with the transition state, followed by the release of the products [24,25]. It plays a functional role via its motion between the two major conformational states, the open and closed conformations. That a balance of structural rigidity and flexibility in the substratebinding loop is essential for enzyme catalysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
