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 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 (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

SUBMITTER: Lakshminarasimhan M 

PROVIDER: S-EPMC2937996 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature