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 (Δ 5 -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 Δ 5 -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 (IC 50 =0.16 ± 0.004 μM) and ethacrynic acid (IC 50 =3.3 ± 0.2 μM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.

Glutathione Transferases as Efficient Ketosteroid Isomerases

“…The k cat values for the isomerase reaction catalyzed by the human HSD3B1 and HSD3B2 enzyme have been reported as 50.2 and 81.5 min −1 (i.e., 0.84 and 1.36 s −1 ). By contrast, the k cat values of the ketosteroid isomerase activity of human, equine, and marmoset GST A3-3 are two orders of magnitude higher, ranging between 204 and 261 s −1 ( Ismail et al, 2021 ).…”

“…By contrast, a porcine enzyme entitled SscGST A2-2, linked to ovarian follicular differentiation and showing 84% sequence identity with the human enzyme, was found to approach the high ketosteroid activity of HsaGST A3-3 ( Fedulova et al, 2010 ). Likewise, CjaGST A3-3 from the common marmoset was shown to have high activity with the steroid substrates ( Ismail et al, 2021 ). In an attempt to find additional GSTs with prominent ketosteroid isomerase activity, mRNAs from the testes of dog, goat, and gray short-tailed opossum were probed for homologs of the horse and human GSTA3 sequences ( Hubert et al, 2020 ).…”

“…CuOOH, cumene hydroperoxide; nonenal, trans -2-nonenal; PEITC, phenethyl isothiocyanate; AD, 5-androsten-3,17-dione. The specific activities are given in units of μmol min −1 mg −1 and compiled from EcaGST A3-3 ( Lindström et al, 2018 ); EcaGST A1-1 (A. Ismail, unpublished); HsaGST A3-3 and HsaGST A1-1 ( Johansson and Mannervik, 2001 ); HsaGST A2-2 ( Johansson and Mannervik, 2001 ; Zhang et al, 2012 ); SscGST A2-2 ( Fedulova et al, 2010 ); SscGST A1-1 ( Fedulova et al, 2011 ); CjaGST A3-3 and CjaGST A1-1 ( Ismail et al, 2021 ); BtaGST A1-1 ( Raffalli-Mathieu et al, 2007 ); CluGST A3-3 (A. Ismail, unpublished); HsaGST A4-4 ( Hubatsch et al, 1998 ).…”

“…Humans would not be investigated until animal data have been obtained. Possibly, the common marmoset could be considered as a model ( Ismail et al, 2021 ).…”

Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species

“…The equine and common marmoset GST A3-3 enzymes are structurally similar to the human enzyme, and they match or exceed the very high steroid isomerase activity of the latter [ 15 , 23 ]. Remarkably, the phylogenetically more closely related GSTA3 homologs from the gonads of domestic pigs ( Sus scrofa ) and cattle ( Bos taurus ) yielded enzymes that differed greatly as ketosteroid isomerases [ 10 , 24 ].…”