Overexpression and mutagenesis of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. Role of cysteines and tyrosines in catalysis.
The overexpression and purification of recombinant rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD; EC 1.1.1.50) in Escherichia coli are described. The properties of the homogeneous recombinant 3 alpha-HSD (r3 alpha-HSD) confirm that a single polypeptide can function as a HSD, as a dihydrodiol dehydrogenase, and as an aromatic aldehyde, ketone, and quinone reductase. Cys-170, Cys-242, and Cys-217, implicated by bromoacetoxysteroid affinity-labeling agents as points of contact for the C-3, C-11, and C-17 positions of steroid ligands, were mutated to alanines. Unexpectedly, the homogeneous C170A and C242A mutants were kinetically similar to wild-type r3 alpha-HSD. By contrast, the C217A mutant gave Km values that were 4-fold higher for androstanedione and 2-fold higher for NADH. Inspection of the recently solved crystal structure of rat liver 3 alpha-HSD (Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., and Lewis, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2517-2521) places Cys-170 and Cys-242 on the periphery of an alpha/beta-barrel so that they cannot be involved in catalysis of steroid recognition. This demonstrates that bromoacetoxysteroid affinity-labeling agents may provide misleading information regarding the topography of steroid hormone binding sites. When NADPH was modeled into the crystal structure of 3 alpha-HSD, Tyr-55 was implicated as the general acid, since it is in close proximity to the C-4 position of the nicotinamide ring and could polarize the substrate carbonyl. In support of this model, the purified Y55F mutant was found to be catalytically inactive, but still formed an E-NADPH complex (measured by fluorescence titration) and an E-NADH-testosterone complex (measured by equilibrium dialysis). The ability of the Y55F mutant to form binary and ternary complexes, but not aid in hydride transfer, is consistent with Tyr-55 acting as the general acid. 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and Tyr-55 is invariant in members of this family where it may perform a similar function. Tyr-205 is present in a pentapeptide sequence that is conserved in HSDs that belong to the short-chain alcohol dehydrogenase family and has been implicated as the general acid within these enzymes. The Y205F mutant was found to be kinetically similar to wild-type r3 alpha-HSD.(ABSTRACT TRUNCATED AT 400 WORDS)
Pawlowski
J E
JE
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Penning
T M
TM
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
D018076:DNA, Complementary; D014443:Tyrosine; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D003545:Cysteine; D007213:Indomethacin