Affinity chromatography of 3-oxosteroid Δ4-Δ5-isomerase of Pseudomonas testosteroni

Benson, Ann M.; Suruda, Anthony; Shaw, Roger; Talalay, Paul

doi:10.1016/0005-2760(74)90245-8

Cited by 30 publications

(9 citation statements)

References 6 publications

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“…3␣-Dehydrogenase [44][45][46]48,52] and 3-ketosteroid-4(5)-isomerase [9,21,47,[53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70] of C. testosteroni ATCC11996 were studied in detail, that we did little experimental examination of corresponding genes in C. testosteroni TA441. Cholic acid and its analogs have an ␣-oriented hydroxyl group at the C-3 position.…”

Section: Discussionmentioning

confidence: 99%

Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)-isomerase and 3α- and 3β-dehydrogenases and evidence for a 100kb steroid degradation gene hot spot

Horinouchi

Kurita

Hayashi

et al. 2010

The Journal of Steroid Biochemistry and Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)-isomerase and 3α- and 3β-dehydrogenases and evidence for a 100kb steroid degradation gene hot spot

Horinouchi

Kurita

Hayashi

et al. 2010

The Journal of Steroid Biochemistry and Molecular Biology

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“…19-Nortestosterone was a gift of Searle Chemical Co., Chicago, IL, and was purified via crystallization and high-vacuum sublimation. KSI was purified to homogeneity, crystallized as previously described (Kawahara et al, 1962;Talalay & Boyer, 1965;Benson et al, 1974;Jarabak et al, 1969), and stored as a suspension in 50% saturated (NH4)2S04 at 4 °C. The enzyme was found to retain almost full activity for at least 3 years.…”

Section: Methodsmentioning

confidence: 99%

Positioning of a spin-labeled substrate analog into the structure of .DELTA.5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and x-ray diffraction methods

et al. 1987

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We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…3-Oxo-A5-steroid isomerase was purified from progesterone-induced Pseudomonas testosteroni following the procedure of Jarabak et al (1969) as modified by Benson et al (1974) except that the affinity chromatography step was performed by using a deoxycholate-ethylenediamine-agarose resin. The species of the enzyme having an isoelectric point of 4.75 was prepared from the mixture of isozymes following the procedure of Ogez et al (1977).…”

Section: Methodsmentioning

confidence: 99%

Proton nuclear magnetic resonance studies of Pseudomonas testosteroni 3-oxo-.DELTA.5-steroid isomerase and its interaction with 17.beta.-estradiol

Benisek¹,

Ogez²

1982

Biochemistry

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Affinity chromatography of 3-oxosteroid Δ4-Δ5-isomerase of Pseudomonas testosteroni

Cited by 30 publications

References 6 publications

Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)-isomerase and 3α- and 3β-dehydrogenases and evidence for a 100kb steroid degradation gene hot spot

Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)-isomerase and 3α- and 3β-dehydrogenases and evidence for a 100kb steroid degradation gene hot spot

Positioning of a spin-labeled substrate analog into the structure of .DELTA.5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and x-ray diffraction methods

Proton nuclear magnetic resonance studies of Pseudomonas testosteroni 3-oxo-.DELTA.5-steroid isomerase and its interaction with 17.beta.-estradiol

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