The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects<sup>*</sup>

Jerussi, Robert A.; Ringold, Howard J.

doi:10.1021/bi00886a028

Cited by 34 publications

(22 citation statements)

References 14 publications

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confidence: 99%

“…The adenine moiety is nested in a hydrophobic pocket formed primarily by the side chains of Val 13 10 VLVVGSGGGALTGAYTAAAQGLTTIVLE 37 (the conserved residues are in bold), in which the third conserved glycine is replaced by alanine (Ala 19 ). These residues are part of the ␤1b-␣1-␤2 portion of the first half of the Rossmann fold.…”

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confidence: 99%

“…However, whereas the 1␣-hydrogen release is an absolute requirement, the 2␤-hydrogen loss is not obligatory (10). A dehydrogenation experiment with the ⌬ 1 -KSTD from Bacillus sphaericus using 2␤-deuterio-5␣-androstane-3,17-dione as substrate showed only 86% loss of the deuterium (10). Although reportedly the deuterated substrate was not the pure 2␤-isomer (10), the incomplete loss of the 2␤-hydrogen could also be due to the formation of a reactive transient species that may undergo fast hydrogen exchange at the C2 atom (9 -11).…”

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confidence: 99%

“…The reactive species was suggested to be either a keto-enolate (10, 11) or a carbanion (9). Both suggestions are based on the fact that ⌬ 1 -KSTD may catalyze hydrogen exchange at the C2 atom of its substrate, even when 1␣-hydrogen expulsion was prevented by the absence of an electron acceptor for the oxidative half-reaction (10,11) or by keeping the flavin coenzyme in the reduced state (9). Indeed, our structures and the observed binding mode of 4-AD show that the active site of the enzyme architecture can stabilize a transient keto-enolate during the reaction, supporting a catalytic mechanism that makes use of keto-enol tautomerization similar to the mechanism of ⌬ 4 -(5␣)-KSTD (32).…”

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Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Rohman

Oosterwijk

Thunnissen

et al. 2013

Journal of Biological Chemistry

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Background: 3-Ketosteroid ⌬ 1 -dehydrogenases catalyze the 1,2-desaturation of 3-ketosteroids. Results: First structures of the enzyme from Rhodococcus erythropolis SQ1, combined with site-directed mutagenesis, clarify its catalytic mechanism. Conclusion: Tyr 487 and Gly 491 promote keto-enol tautomerization, whereas Tyr 318 /Tyr 119 and FAD abstract a proton and hydride ion, respectively. Significance: This study is an important step toward tailoring the enzyme for steroid biotransformation applications.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Rohman

Oosterwijk

Thunnissen

et al. 2013

Journal of Biological Chemistry

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“…In contrast, the optimal pH varied depending on the method used and was higher with (pH 9) than without (pH 8) dye. As suggested by Jerussi and Ringold (22), an apparent increase in dehydrogenase velocities at higher pHs is expected to reflect more the ionization state of the Wurster's blue than that of the active site of the enzyme. Efficiencies of various electron acceptors typical of flavoprotein-catalyzed reactions were compared by HPLC quantification of the ⌬ 1,4 derivative produced from ⌬ 1 -androstene-3,17-dione.…”

Section: Vol 178 1996mentioning

confidence: 81%

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization

Florin¹,

Köhler²,

Grandguillot³

et al. 1996

J Bacteriol

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Comamonas testosteroni ⌬Comamonas (formerly Pseudomonas) testosteroni is a soil microorganism able to grow at the expense of testosterone, progesterone, and various bile acids as unique sources of carbon and energy. Complete assimilation of these substrates is achieved through a complex metabolic pathway involving many enzymatic steps of oxidation responsible for the breakdown of the steroid nucleus (9,25,29). The introduction of double bonds into ring A at the early stage of this catabolic process is critical since it initiates the molecule cleavage. Upon induction with testosterone, C. testosteroni synthesizes three different dehydrogenases able to promote the desaturation of ring A of 3-ketosteroids: one (Fig. 1). All three enzymes have properties specific to flavoproteins, but only ⌬ 4 (5ß)DH was purified and characterized as being a flavin adenine nucleotide (FAD)-dependent enzyme (10,30).In a previous work, we isolated and characterized the gene encoding ⌬ 1 DH from C. testosteroni ATCC 17410 (37). A Tn5-insertion mutant (named 06) defective in testosterone utilization was found to express very low levels of both ⌬ 1 -and ⌬ 4 (5␣)DH activities while producing other quantifiable steroid-modifying enzymes at parental levels. These results strongly suggested that the genes coding for ⌬ 1 DH and ⌬ 4 (5␣)DH belong to the same transcription unit. In the present study, we demonstrate that the two genes are adjacent on the chromosome of C. testosteroni. The putative ⌬ 4 (5␣)DH peptide deduced from the gene sequence showed weak homology with the ⌬ 1 DH protein except in the N terminus, where a highly conserved FAD-binding site is present. These findings indicate that both dehydrogenases, though having closely related enzymatic activities, are probably not derived from a common ancestor. MATERIALS AND METHODSBacterial strains, phages, and plasmids. Bacterial strains, phages, and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli and Pseudomonas putida were grown routinely in LB broth (38) or on Mueller-Hinton agar plates (Pasteur Diagnostics, France). When necessary, the following antibiotics were added to growth media: ampicillin (50 g/ml), chloramphenicol (20 g/ml), and tetracycline (12.5 g/ml) for E. coli, and chloramphenicol (300 g/ml) for P. putida. C. testosteroni was grown in YE broth as described previously (37). E. coli XL-1 Blue, used for single-stranded DNA production, was cultured in nutrient broth composed of (per liter) Bacto Tryptone (35 g), Bacto Yeast Extract (20 g; Oxoid Ltd., Basingtoke, England), and NaCl (5 g) at pH 7.5. All bacterial cultures were incubated at 30ЊC with shaking.Biotransformation of steroids. Recombinant bacteria harboring DNA fragments from C. testosteroni were assayed for the expression of various enzyme activities of the steroid catabolic pathway (see below). Enzyme-catalyzed conversion of steroids into specific metabolites was demonstrated by thin-layer

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Immobilisierung von corynebakterium simplex in thioliertem polyvinylalkohol zur mikrobiologischen steroidumwandlung

Manecke

1982

Angew. Makromol. Chem.

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ZUSAMMENFASSUNG:Polyvinylalkohol wurde durch polymeranaloge Reaktion mit 3-Mercaptopropionsaure zu einem zum EinschluB von Mikroorganismen geeigneten Polymeren derivatisiert.Die SUMMARY:Whole cells of Arthrobacter simplex (A.T.C.C. 6946) were entrapped in poly(viny1 alcohol) which was previously esterified with 3-mercaptopropionic acid.The entrapped microorganisms showed steroid-Al-dehydrogenase activity and the transformation of hydrocortisone to prednisolone was investigated. The steroid-Aldehydrogenase activity of the immobilized cells increased in the presence of an inducer such as hydrocortisone. The effect of cell-, substrat-and cofactorconcentration, pH, and temperature was determined. The half-live was analyzed with and without nutrients.The activity of the immobilized Arthrobacter simplex product was 200 prnol-gh-'

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The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects^*

Cited by 34 publications

References 14 publications

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization

Immobilisierung von corynebakterium simplex in thioliertem polyvinylalkohol zur mikrobiologischen steroidumwandlung

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The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects*

Cited by 34 publications

References 14 publications

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization

Immobilisierung von corynebakterium simplex in thioliertem polyvinylalkohol zur mikrobiologischen steroidumwandlung

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The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects^*