Figure 1.
The mechanism of the 3α-hydroxysteroid dehydrogenase/carbonyl reductase-catalyzed reaction.
3α-HSD/CR reversibly catalyzes the oxidation of androsterone with NAD+ to form androstanedione with NADH in a sequential order bi bi kinetic mechanism with NAD+ added first and NADH released last. (a) The chemical step wherein a tetrad of catalytically important N86, S114, Y155 and K159 residues in the active site are shown. Y155 acts as a general base to facilitate the hydride transfer from the 3β-hydrogen of androsterone to the nicotinamide ring. S114 interacting with P185 maintains the conformation of the substrate-binding loop, in which T188 binds with NAD+. (b) The rate-limiting step for NADH and proton release. Protons are released through the proton relay system, via Y155, K159 and N86, to the solvent.
Figure 2.
Structural alignment of P.s. 3α-hydroxysteroid dehydrogenase and C.t. 3α-HSD/CR.
A. Coenzyme NADH binding to P.s. 3α-HSD (yellow; pdb:2dkn) induces the loop-helix transition (blue) and an unresolved flexible loop (black dotted line) between P191 and F209 for the NAD+ bound C.t. 3α-HSD/CR (green; pdb:1fk8). NAD+ and key residues in the active site of 3α-HSD/CR are also labeled. (B) Close-up of the interaction of the substrate-binding loop with NAD+. The interactions of P185 at the hinge region with both S114 and nicotinamide ring, and T188 in the substrate-binding loop with the amide NH of NAD+ are shown as blue dotted lines. The distances from P185 to the nicotinamide ring of NAD+ and the hydroxyl group of T188 with the amide NH of NAD+ are 4.1 and 3.6 Å, respectively. The image was generated using the PyMOL program.
Figure 3.
CD spectra of wild-type 3α-HSD/CR and mutant enzymes.
The CD spectra of wild-type and the P185A, P185G, T188A and T188S mutant enzymes. The mutation at P185 causes an increase in the intensity at 222 nm, compared to that of the wild-type enzyme. The CD spectra were measured at 8.8 µM enzyme in 10 mM phosphate at pH 7.5 at room temperature.
Figure 4.
The fluorescence emission spectra of wild-type 3α-HSD/CR and its mutants.
The fluorescence spectra of wild-type and P185A, P185G, T188A, T188S, W173F/P185W, and W173F/T188W mutant enzymes. The mutants of W173F/P185W and W173F/T188W display a red shift in the maximum wavelength at 345 and 349 nm, respectively. The protein fluorescence spectra were measured at 2 µM enzyme in 40 mM Hepes at pH 7.5 at room temperature. The excitation wavelength is 295 nm and the emission range was recorded from 300 to 450 nm.
Figure 5.
The fluorescence titration spectra of 3α-HSD/CR and its mutants.
A. The fluorescence titration spectra of the mutant enzymes of P185A, P185G, T188A and T188S for increased addition of NADH. The incremental addition of NADH gradually causes a blue shift at the maximum wavelength for the P185A and P185G mutants. For clarity, only 0(black), 14(red), 55(green) and 97(yellow) µM NADH titrations are shown. B. The fluorescence titration curves by varying the concentrations of NADH for the wild-type and P185A, P185G, T188A and T188S mutant enzymes. Corrected for the inner filter effect, the difference (ΔF) in the intrinsic protein fluorescence titrated by NADH is shown. The lines represent the fit of the data points of the wild-type and mutants to Equation 11. The data for the wild-type enzyme is from Ref. 16.
Table 1.
The kinetic constants and isotope effects for wild-type and mutant 3α-HSD/CRa.
Figure 6.
Pre-steady-state kinetics of 3α-HSD/CRs.
The stopped-flow progress curves for 3α-HSD/CRs catalysis of the reaction of the undeuterated (blue diamond) and deuterated (black circle) androsterone with NAD+. Reactions were performed in 1 µM WT, 1 mM NAD+, 17.5 µM undeuterated or deuterated androsterone at 0.1 M Caps, pH 10.5. The line represents the fit of the data points to Equation 10, giving an apparent value for kobs of 309 s−1 for the reaction catalyzed by 3α-HSD/CRs with the deuterated androsterone.
Table 2.
Specificity constants and free energy differences in ground state and transition state energies between wild-type and mutant 3α-HSD/CRsa.
Figure 7.
Superposition of the NAD+ bound binary complex (1fk8) with the molecular modeled structures of apo- and holoenzyme.
(A) Ribbon diagram of the crystal structures of 3α-HSD/CR (grey) and the modeled structures of apo- (green) and holoenzyme (purple). The conformational changes in the inserted flexible substrate binding loops are shown in gold and blue for apo- and holoenzyme, respectively. NAD+ from the crystal structure and the modeled binary complex is shown as a line with yellow and red colors, respectively. (b) Close-up view of the potential steric hindrance in residues A187, T188, and P200 (grey line) of apoenzyme with NAD+ molecule in a holoenzyme model. Close contacts is not possible in the structure without causing conformational changes in the loop. T188 and T190 form hydrogen bonds (green line) with nicotinamide of NAD+ in the modeled holoenzyme.
Figure 8.
Free energy profiles for catalysis by wild-type and mutant 3α-HSD/CRs.
The free energy differences calculated from the data in Table 2 are normalized to the free energy of the ground state (E+NAD+S), containing the enzyme of wild-type and mutants (E), and the free substrates, NAD+ and androsterone (S). Energy levels are shown for the ground binding energy of the enzyme-NAD+ complex (E-NAD+S), and the transition state (TS) energy for the hydride transfer from androsterone to NAD+ in the reaction catalyzed by the wild-type and mutant 3α-HSD/CRs, respectively. The free energy profiles illustrate the differential destabilization of the ground state and the transition state caused by the mutation at P185 and T188 in the 3α-HSD/CR catalyzed reaction, as described in the text.