Abstract
3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.
Highlights
K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively
To shed more light on the conserved catalytic residues in 3␣-HSD/CR and the short chain dehydrogenase/reductase (SDR) family, using mutagenesis and kinetic studies we investigated the roles of the triad of Ser-114, Tyr-155, and Lys-159 in the catalytic mechanism of 3␣-HSD/ CR
Structure analysis, site-directed mutagenesis, and kinetic characterization elucidate the role of the catalytic triad
Summary
5Ј-gccgtcgtcatctcggccgtggcttccgcgc-3Ј 5Ј-gcgcggaagccacggccgagatgacgacggc-3Ј 5Ј-gggcggaaatctggcctttgcgggcagcaagaatg-3Ј 5Ј-cattcttgctgcccgcaaaggccagatttccgccc-3Ј 5Ј-ctggcctatgcgggcagcgcgaatgctttgacggtggc-3Ј 5Ј-gccaccgtcaaagcattcgcgctacccgcataggccag-3Ј 5Ј-gggcggaaatctggcctttgcgggcagcaagaatg-3Ј 5Ј-cattcttgctgcccgcaaaggccagatttccgccc-3Ј 5Ј-cggaaatctggcctttgcgggcagcgcgaatgctttgacgg-3Ј 5Ј-ccgtcaaagcattcgcgctgcccgcaaaggccagatttccg-3Ј a The boldface codons indicate the mutation on the amino acid residues by the replacement of the underlined codons. b Plasmid S114A/Y155F mutant was constructed with S114A plasmid and Y155F primer. The structures of the binary complex with NADϩ cofactor and apoenzyme of 3␣-HSD/CR have been solved [3, 17]. The NADϩ cofactor is bound at the C-terminal ends of the -strands in the 3␣-HSD/CR from C. testosterone. The chemical mechanism for the 3␣-HSD/ CR-catalyzed reaction is based on sequence comparisons and structure analyses in the SDR family [17]. A triad of Ser-114, Tyr-155, and Lys-159 in 3␣-HSD/CR is conserved within the SDR family. The functional role of Ser-114, Tyr-155, and Lys-159 in the 3␣-HSD/CR-catalyzed reaction, has not been analyzed in detail kinetically. To shed more light on the conserved catalytic residues in 3␣-HSD/CR and the SDR family, using mutagenesis and kinetic studies we investigated the roles of the triad of Ser-114, Tyr-155, and Lys-159 in the catalytic mechanism of 3␣-HSD/ CR. The acidbase chemistry was studied through pH profiling to evaluate the residues involved in the general base to catalysis
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