Ketosteroid

Abstract: Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testos...

Abstract: Publisher Summary Stereospecific and reversible interconversions of hydroxyl and ketone functions of steroids are catalyzed by pyridine nucleotide-dependent hydroxysteroid dehydrogenases. Enzymes of this class are widely distributed among microorganisms and in animal tissues. Discrete catalytic proteins are concerned with reversible oxidations at specific locations, i.e., of 3 α -, 3 β -, ll β , 17 β -, 20 α -, and 20 β -hydroxysteroids. This chapter describes the assay method, purification procedure, and properties of hydroxysteroid dehydrogenases of Pseudomonas testosterone . 3 α -hydroxysteroid dehydrogenase ( α -enzyme) and (3 and 17) β -hydroxysteroid dehydrogenase ( β -enzyme) are two steroid-induced hydroxysteroid dehydrogenases that are isolated from Pseudomonas testosteroni , a soil microorganism which can utilize testosterone, or related steroid hormones, as the only source of organic carbon. Both α - and β -enzymes are inactivated by low concentrations of heavy metal ions and p -chloromercuribenzoate. Both enzymes are protected against inactivation by DPN. β -Enzyme is powerfully stabilized by low concentrations of estradiol-17 β . The asssay method, purification procedure, and properties of 20 β -hydroxysteroid dehydrogenase of Streptornyces hydrogenmas is described in the chapter.

Abstract: In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Δ1-dehydrogenase and 3-ketosteroid-Δ4(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.