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J. Biol. Chem., Vol. 280, Issue 38, 32827-32834, September 23, 2005

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Mechanistic Insights into the Isochorismate Pyruvate Lyase Activity of the Catalytically Promiscuous PchB from Combinatorial Mutagenesis and Selection^*

From the Laboratorium für Organische Chemie, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg⁵⁴, Glu⁹⁰, Gln⁹¹) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg⁵⁴ was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu⁹⁰ in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu⁹¹ in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Received for publication, June 24, 2005

^* This work was supported by the Swiss Federal Institute of Technology (ETH) Zürich and the Schweizerischer Nationalfonds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratorium für Organische Chemie, Swiss Federal Inst. of Technology Zürich, ETH Hönggerberg—HCI F333, CH-8093 Zürich, Switzerland. Tel.: 41-44-632-2908; Fax: 41-44-633-1326; E-mail: kast{at}org.chem.ethz.ch.

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