Enzyme-catalyzed allylic rearrangements

Schwab, John M.; Henderson, Barry

doi:10.1021/cr00105a007

Cited by 95 publications

(85 citation statements)

References 157 publications

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“…A llylic rearrangements play a fundamental role in the biosynthesis of terpenes and steroid hormones and in the biodegradation of fatty acids (1). The enzymatic rearrangement of ␤-␥ unsaturated ketones requires a general base, usually a carboxylate, to abstract the ␣-proton of the ketone ( Fig.…”

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Structural evidence for a programmed general base in the active site of a catalytic antibody

Golinelli‐Pimpaneau

Gonçalves

Dintinger

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation-interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.A llylic rearrangements play a fundamental role in the biosynthesis of terpenes and steroid hormones and in the biodegradation of fatty acids (1). The enzymatic rearrangement of ␤-␥ unsaturated ketones requires a general base, usually a carboxylate, to abstract the ␣-proton of the ketone ( Fig. 1; refs. 1 and 2). This reaction leads to a dienol or dienolate high-energy intermediate 3 that does not possess a charge complementing that of the carboxylate. Therefore, antibodies elicited against a hapten that mimics the transition state or the dienol intermediate of allylic rearrangement would acquire a properly positioned general base carboxylate to catalyze this reaction only by serendipity. However, the extensive experience available on catalytic antibodies (3, 4) and the structures of catalytic antibodies elicited by transition state analogue haptens show that properly positioned chemically reactive residues are rarely present in the active site (5). An alternative approach that aims to generate functional residues, also termed ''bait and switch'' (6, 7), uses a charged hapten to induce the required complementary charged residue (8, 9). Haptens containing a positive charge have provided antibody catalysts for important reactions such as acyl transfer (7), elimination (9, 10), and phosphodiester hydrolysis (11). However, in the absence of structural data, it is not possible to establish unambiguously the nature and identity of the catalytic residue that has been induced and the relationship between the location of the haptenic charge and the position of the catalytic residue in the antibody combining site.Indeed, in the few cases in which the use of a hapten containing a positively charged moiety successfully induced catalytic antibodies and in which the structure of the hapten-antibody complex was determined, there was no negatively charged residue in the active site directly facing the positive charge, but stabilization of the haptenic charge was mediated mostly by cation-interactions (12-14). Herein, we report the structure, at 1.87-Å resolution, of the complex of an antibody catalyzing an allylic rearrangement with its cationic hapten. We provide direct ...

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confidence: 99%

Structural evidence for a programmed general base in the active site of a catalytic antibody

Golinelli‐Pimpaneau

Gonçalves

Dintinger

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…5 -3-Ketosteroid isomerase (KSI) catalyzes the allylic isomerization of the 5,6 double bond of ⌬ 5 -3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton at a rate approaching the diffusion limit (23,26). This reaction is part of the steroid hormone biosynthetic pathway from cholesterol in animals and is part of the biodegradative pathway for steroids in bacteria such as Comamonas testosteroni, formerly known as Pseudomonas testosteroni (28), and Pseudomonas putida biotype B, which can live on steroids as a sole source of carbon.…”

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Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

et al. 1997

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In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by sitedirected mutagenesis. All cysteine mutations caused only a slight decrease in the k cat value, with no significant change of K m for the substrate. Even modification of the sulfhydryl group with 5,5-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an activesite-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by sitedirected mutagenesis. For D103A mutant KSI there was a significant decrease in the k cat value: the k cat of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k cat value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.

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“…The reaction involves a stereospecific intramolecular transfer of a proton by cleaving a proton bonded to a carbon atom at the 4␤ position of the steroid ring. This enzymatic reaction has been of considerable interest since the removal of a proton from one point in the substrate and readdition of the proton by intramolecular transfer are an elementary process in the mechanism of various enzymatic reactions (21). Two isomerases catalyzing this enzymatic reaction have been found in soil bacteria, Comamonas testosteroni (previously known as Pseudomonas testosteroni) and Pseudomonas putida biotype B, which can live on the steroid as a sole carbon source.…”

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“…KSI from C. testosteroni has been studied very extensively, and its catalytic mechanism has been elucidated at the molecular level (19,21). The roles of active site residues in the KSI of C. testosteroni have been characterized well by affinity and photoaffinity labeling studies (1,6,9,18) and especially by site-directed mutagenesis of the specific amino acid residues in the enzyme utilizing the gene which was cloned and sequenced (4,5,13).…”

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Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Kim

Choi

1995

J Bacteriol

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Enzyme-catalyzed allylic rearrangements

Cited by 95 publications

References 157 publications

Structural evidence for a programmed general base in the active site of a catalytic antibody

Structural evidence for a programmed general base in the active site of a catalytic antibody

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

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