On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

Chao, Lin; Hoffman, Timothy Z.; Wirsching, Peter; Barbas, Carlos F.; Janda, Kim D.; Lerner, Richard A.

doi:10.1073/pnas.94.22.11773

Cited by 18 publications

(10 citation statements)

References 32 publications

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“…Indeed, these methods allow the use of a hapten that does not need to match precisely the transition state of the reaction but rather elicits a strategically positioned functional residue. Therefore, antibodies resulting from these strategies have been found to catalyze different reactions that use the same mechanism, provided that the various substrates possess the common chemical function with which the induced residue can react (10,36,37). This finding is also illustrated by antibody 4B2, which catalyzes both the Kemp elimination (20) and the allylic rearrangement of a ␤-␥ unsaturated ketone.…”

Section: Discussionmentioning

confidence: 58%

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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Section: Discussionmentioning

confidence: 58%

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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“…It has been established that the lysine's amine attack on the carbonyl group of various substrates (including the immunizing hapten) leads to the formation of a covalent enamine intermediate (Scheme 1). Consequently, a variety of reactions that proceed via an enamine intermediate are catalyzed by these antibodies: aldol and retro‐aldols (Wagner et al 1995; Barbas et al 1997), decarboxylation of β‐keto acids (Bjornestedt et al 1996), and an allylic rearrangement (Lin et al 1997).…”

Section: Resultsmentioning

confidence: 99%

Catalytic and binding poly‐reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution

James

Tawfik

2001

Protein Science

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It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit some activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins-serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities-in the shape of the cavities and the position of catalytic lysine residues-both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active-site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind- inefficient and rudimentary as they are-can provide a considerable selective advantage and a starting point for the evolution of new functions.

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“…Catalytic antibodies have proven utility for chemical catalysis in vitro (1)(2)(3)(4)(5)(6)(7). In living cells, these protein catalysts could accomplish a variety of reactions to modify cell behavior or generate reporter molecules for biological assays.…”

mentioning

confidence: 99%

A Catalytic Antibody Produces Fluorescent Tracers of Gap Junction Communication in Living Cells

Subauste¹,

List²,

Guan³

et al. 2001

Journal of Biological Chemistry

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On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

Cited by 18 publications

References 32 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

Catalytic and binding poly‐reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution

A Catalytic Antibody Produces Fluorescent Tracers of Gap Junction Communication in Living Cells

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