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

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 sitedirected 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 (Cys 101 ) that is required for catalysis and interacts with Asp 17 , Thr 102 , and an ordered water molecule in the active site. Asp 17 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 Cys 101 and Asp 17 , Thr 102 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-␣ root mean square deviation), structural differences in the vicinity of Cys 101 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.

A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans

“…Therefore, these two oxygen atoms were conservatively modeled as water molecules in the crystal structure. We speculate that photoreduction and subsequent redox chemistry caused by synchrotron radiation may be responsible for this modification, as noted previously in Pseudomonas fluorescens isocyanide hydratase (46). In total, the structural features of the Glx3 active site are consistent with a hydrolase employing a Cys-His-Glu catalytic triad with a reactive cysteine that attacks small electrophilic substrates.…”

“…3C), consistent with a role in detoxifying low molecular mass glyoxals. As observed in many other DJ-1 superfamily crystal structures (20,(45)(46)(47)(48)(49), the active site cysteine residue is surrounded by unusual electron density indicative of probable partial oxidation of the reactive thiol(ate). Two oxygen atoms are modeled at 2.25 and 2.06 Å from the S␥ atom of Cys 136 (Fig.…”

Short Carboxylic Acid–Carboxylate Hydrogen Bonds Can Have Fully Localized Protons

“…Hexahistidine-tagged human DJ-1 and E. coli YajL were expressed from pET15b constructs in BL21(DE3) E. col, purified using Ni 2+ -metal affinity chromatography, and thrombin-cleaved to remove the histidine tag as described previously 23, 24 . The hexahistidine tag was removed by thrombin cleavage and the final protein contains the vector-derived sequence GSH- before the first methionine in the native protein sequence.…”

Influence of Peptide Dipoles and Hydrogen Bonds on Reactive Cysteine PKa Values in Fission Yeast DJ-1

“…The expressed protein bears a thrombin-cleavable Nterminal hexahistidine tag to facilitate purification by metal affinity chromatography. Cells growth, protein expression, and purification were performed as described previously [16]. The purified protein was concentrated to 21 mg/ml in storage buffer (25 mM HEPES pH=7.5, 100 mM KCl, 2 mM DTT) using stirred pressure cell and centrifugal concentrators with 10 kDa nominal molecular weight cutoffs.…”