Identifying Androsterone (ADT) as a Cognate Substrate for Human Dehydroepiandrosterone Sulfotransferase (DHEA-ST) Important for Steroid Homeostasis: STRUCTURE OF THE ENZYME-ADT COMPLEX

Ho-Jin Chang,Rong Shi,Peter Rehse,Sheng-Xiang Lin

doi:10.1074/jbc.m310446200

Ho-Jin Chang, Rong Shi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m310446200

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Abstract

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 A resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.

Highlights

Human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3؅-phosphoadenosine-5؅-phosphosulfate to dehydroepiandrosterone (DHEA)
It is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA
The structure of human DHEA-ST in complex with ADT has been solved at 2.7 Å resolution, confirming ADT recognition

Summary

EXPERIMENTAL PROCEDURES

Materials—DHEA, ADT, and PAPS were obtained from Sigma Chemical Co. (9,11-3H (N))-ADT (54 Ci/mmol) and (4-14C)-DHEA (53 mCi/mmol) were purchased from PerkinElmer Life Sciences. DHEA-ST activity was assayed at 37 °C at various time intervals in a final reaction volume of 150 ␮l containing 20 mM Tris, pH 7.5, 15 mM MgCl2, 50 ␮M PAPS, 2% ethanol, and various amounts of steroids. We used the refined 2.5 Å resolution structure of the human DHEA-ST in complex with DHEA (accession code 1J99 in Protein Data Bank) as the starting model. After the structure was rebuilt and refined by several cycles, the electron density corresponding to ADT and sulfate group was clear enough to put the two molecules in the substrate binding site and the cofactor-binding site respectively. Structural comparison studies using the ADT complex, the DHEA complex, the PAP complex (accession code 1EFH in Protein Data Bank), and the estrogen sulfotransferase (EST) structure (accession code 1AQU in Protein Data Bank) were performed using the lsq routines of the O program [23]

RESULTS AND DISCUSSION

ADT DHEA

Hydrogen bonds