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PubMed1995

Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-delta 1-dehydrogenase, 3-ketosteroid-delta 5-isomerase and a hypothetical regulatory protein.

The Arthrobacter simplex gene coding for 3-ketosteroid-delta 1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-delta 1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-delta 1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.

Amino Acid Sequence↗

PubMed1982

Inactivation of delta5-3-ketosteroid isomerase(s) from beef adrenal cortex by beta, gamma-acetylenic ketosteroids.

The beta, gamma-acetylenic ketosteroids, 5-10-seco-19-norpregn-5-yne-3,-10,20-trione approximately 1 and 5,10-secoestr-5-yne-3,10,17-trione approximately 2 irreversibly inactivate both the C19-and the C21-delta 5-3-ketosteroid isomerase activities of beef adrenal cortex microsomes. At saturating concentrations of inhibitor half-lives of these enzyme activities vary from 45 to 240 s. It is uncertain whether the enzyme generates its own alkylating agent by isomerizing compounds approximately 3 and approximately 4 to the corresponding allenic ketones, namely (4R)-5,10-seco-19-norpregn-4,5-diene-3,10,20-trione approximately 3 and (4R)-5,10-secoestra-4,5-diene-3,10,17-trione approximately 4 since these are formed spontaneously in the buffer used to stabilize enzyme activity. In the presence of catalytic quantities of adrenal enzyme compound approximately 4 is a powerful competitive inhibitor for both 5-androstene-3,17-dione (Ki 8.0 microM) and 5-pregnene-3,20-dione (Ki 3.5 microM) indicating that the eventual alkylating event is active site-directed. The differences in Ki values and half-lives for inactivation support the view that the C19- and C21-delta 5-3-ketosteroid isomerase activities do not reside at the same catalytic site in beef adrenal cortex microsomes.

Adrenal Cortex↗

PubMed1995

Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase.

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1975

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

The photoinactivation of the Δ (5)-3-ketosteroid isomerase of Pseudomonas testosteroni in the presence of 3-oxo-4-estren-17β-yl acetate and air is accompanied by destruction of histidine and aspartate (or asparagine). The first order rate constant of photoinactivation of the enzyme is equal, within experimental error, to the first order rate constant for the destruction of a single aspartate (or asparagine) residue and is considerably greater than the first order rate constant for the destruction of a single residue of histidine. When the photolysis is carried out under anaerobic conditions, only aspartic acid (or asparagine) is destroyed as enzyme is inactivated. Both inactivation and aspartate (or asparagine) destruction occur to a greater extent in the absence of oxygen than in its presence. The destruction of histidine, on the other hand, is found to be strictly oxygen-dependent. These results suggest that photochemical modification of a single residue of aspartate (or asparagine) is largely, if not entirely, responsible for photoinactivation of the enzyme under these conditions. When irradiated in the presence of 3-oxo-4-entren-17β-yl acetate, performic acid-oxidized bovine pancreatic ribonuclease does not suffer any detectable destruction of its aspartic or asparaginyl residues but does undergo significant destruction of its histidine residues. These observations suggest that the aspartate (or asparagine) residue modification found with isomerase is an active site-directed photochemical reaction, whereas the modification of histidine may not be.

Amino Acids↗

PubMed1963