Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

Authors

Mahadevan Lakshminarasimhan, University of Nebraska - Lincoln
Peter Madzelan, University of Nebraska LincolnFollow
Ruth Nan, Washington University in St. Louis
Nicole Marie Milkovic, University of Nebraska - Lincoln
Mark A. Wilson, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

2010

Citation

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 38, pp. 29651–29661, September 17, 2010

Comments

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Abstract

Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys101) that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active site. Asp¹⁷ has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys¹⁰¹ and Asp¹⁷, Thr¹⁰² is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 Å C-α root mean square deviation), structural differences in the vicinity of Cys¹⁰¹ disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.