Marmoset glutathione transferases with ketosteroid isomerase activity

Abstract

The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5-androsten-3,17-dione (Δ⁵-AD) of 62.1 ± 1.8 μmol min^-1 mg^-1, and a k_cat value of 261 ± 49 s^-1. The second ketosteroid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Δ⁵-AD and a 37-fold lower k_cat value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC₅₀=0.16 ± 0.004 μM) and ethacrynic acid (IC₅₀=3.3 ± 0.2 μM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.

Keywords: 1-chloro-2,4-dinitrobenzene, (CDNB); 4-androsten-3,17-dione, (Δ4-AD); 5-Androsten-3,17-dione; 5-Pregnen-3,20-dione; 5-androsten-3,17-dione, (Δ5-AD); 5-pregnen-3,20-dione, (Δ5-PD); Alpha glutathione transferase; CjaGST A1-1; CjaGST A3-3; Glutathione transferase, (GST); Glutathione, (GSH); SDS-PAGE, (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); Steroid hormone synthesis; allyl isothiocyanate, (AITC); phenethyl isothiocyanate, (PEITC).

Fig. 1

Structure of glutathione, L-γ-glutamyl-L-cysteinyl-glycine.

Fig. 2

Double-bond isomerization catalyzed by GSTs with ketosteroid isomerase activity. The reaction with 5-adrosten-3,17-dione is analogous to the 5-pregnen-3,20-dione isomerization shown. The thiolate of glutathione serves as a base removing a C4 proton, and the steroid is then reprotonated via the active-site Tyr9.

Fig. 3

Alignment of the amino acid sequences of the Alpha class enzymes CjaGST A1-1, CjaGST A3-3, HsaGST A3-3, and EcaGST A3-3. G-site residues are indicated in yellow, while H-site residues are indicated in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Active-site models of CjaGST A1-1 (light blue) and CjaGST A3-3 (pink) based on the crystal structure of HsaGST A3-3 in complex with 4-androsten-3,17-dione. The steroid (colored by element) is rendered in ball and stick. The functional thiol of glutathione (yellow) and Tyr9 are shown in the lower left corner, adjacent to the steroid. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Dependence of initial rates of double-bond isomerization for the two marmoset GSTs. Measurements were made in triplicate in the standard assay system with different concentrations of Δ⁵-AD and a fixed concentration of 1.0 mM glutathione.

Fig. 6

Inhibition of CjaGST A3-3 by the organotin compound tributyltin acetate (A) and the diuretic drug ethacrynic acid (B). Remaining enzyme activity measured with CDNB as substrate in the standard assay system was determined at different inhibitor concentrations. The measurements were carried out in triplicate and the data were subjected to nonlinear regression analysis.