A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

Micah T. Nelp,Yang Song,Vicki H. Wysocki,Vahe Bandarian

doi:10.1074/jbc.m115.704791

Abstract

Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using(18)O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles.

Highlights

Nitrile hydratases (NHases)2 catalyze the formation of amides from nitriles in aqueous, pH-neutral environments without any carboxylic acid byproducts [1, 2]
To find direct evidence of either a protein-based or a solventbased nucleophile, we became interested in the observations made by Heinrich et al [14] with an iron NHase mimic bearing two sulfenato ligands that is capable of hydrating nitriles with up to 50 turnovers
Toyocamycin nitrile hydratase (TNH) is a cobalt-type nitrile hydratase, encoded by the toyJKL genes of Streptomyces rimosus, which catalyzes the hydration of toyocamycin to sangivamycin [19]

Summary

16OH NH

The modified cysteine ligands of the active site metal complex [4, 15]. The main difference between ToyJ and the full complement of ToyJKL is that ToyJ alone has lower catalytic efficiency (kcat/Km) and specificity for toyocamycin, suggesting that the ␤-subunits generally, or the ␤- and ␥-subunits in TNHase, are responsible for the substrate specificity and increased catalytic efficiency of these enzymes [16]. We have carried out 18O incorporation experiments using H218O and ToyJ, which unambiguously show that the source of the product amide oxygen is a non-exchangeable ToyJ-bound oxygen that, when taken in the context of other studies of NHases (4 – 6), is likely the nucleophilic oxygen of the cysteine-sulfenate

Experimental Procedures

Results and Discussion

Processed Data

Minimum Error

18OH S