Role of S114 in the NADH-induced conformational change and catalysis of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. In this study, we characterize the role of the conserved residue S114 in cofactor binding and catalysis, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching and anisotropy measurements. The catalytic efficiency of V/K(NADH)Et for wild-type and S114A is 1.5 x10(7) and 3.8 x 10(3) M(-1) s(-1), respectively, suggesting that NADH association to wild-type and S114A mutant enzymes involves two steps, a bimolecular binding step and isomerization. The binding of NADH into a hydrophobic pocket in the active site of wild-type and S114A mutant enzymes restricts its motion and shields the fluorescence quenching from solvent, with an increase in the fluorescence intensity and a blue shift at the maximum wavelength. Furthermore, the binding of NADH leads to the protein fluorescence quenching, mainly due to fluorescence resonance energy transfer to NADH. S114A mutant enzyme decreases 3100-fold in V/Et with no apparent change in K(m) for substrates. Addition of NADH to S114A mutant enzyme induces a secondary structural change. These results suggest that S114 is important to maintain the correct conformation for the nucleotide binding and facilitate the reaction. Substitution of alanine for S114 eliminates the hydrogen bonding interaction with P185, causing a conformational change in a nonproductive binding of NADH and a significant loss of activity.
Chang
Yi-Hsun
YH
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80731, Taiwan.
Huang
Tzu-Jung
TJ
Chuang
Lea-Yea
LY
Hwang
Chi-Ching
CC
D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D005454:Fluorescence Polarization; D057927:Hydrophobic and Hydrophilic Interactions; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D009243:NAD; C425306:3alpha-hydroxysteroid dehydrogenase-carbonyl reductase; D006913:Hydroxysteroid Dehydrogenases