Evolution of new enzymatic function by structural modulation of cysteine reactivity in Pseudomonas fluorescens isocyanide hydratase.

The Journal of Biological Chemistry
Mahadevan LakshminarasimhanMark A Wilson

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 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-α 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 b...Continue Reading

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Citations

Aug 4, 2015·Protein Science : a Publication of the Protein Society·Nicole M MilkovicMark A Wilson
Nov 8, 2011·The Biochemical Journal·Aleksander W RoszakRichard J Cogdell
Jan 10, 2017·Nature Chemical Biology·Bettina M NestlBernhard Hauer
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Jul 6, 2019·Journal of Synchrotron Radiation·Henry van den Bedem, Mark A Wilson
Dec 6, 2019·Proceedings of the National Academy of Sciences of the United States of America·Medhanjali DasguptaMark A Wilson
Oct 9, 2013·Acta Crystallographica. Section D, Biological Crystallography·Fabienne FurtMark A Wilson
Apr 6, 2021·Archives of Biochemistry and Biophysics·Esha SircarRajib Sengupta

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