Abstract
The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.
Highlights
Nitrile hydratases (NHases, EC 4.2.1.84) are metalloenzymes that catalyze the hydration of nitriles to their corresponding amides under ambient conditions and physiological pH [1,2]
NHases have attracted substantial interest as biocatalysts in preparative organic chemistry, as they can hydrate a wide range of synthetic nitrile substrates, resulting in their exploitation for biotechnological purposes as biocatalysts in the production of acrylamide and nicotinamide [4]
NHases are useful in the bioremediation of chemical and wastewater runoff, for the hydration of nitrile-based pesticides such as bromoxynil, and are becoming increasingly recognized as biocatalysts for green chemical processes [6]
Summary
Nitrile hydratases (NHases, EC 4.2.1.84) are metalloenzymes that catalyze the hydration of nitriles to their corresponding amides under ambient conditions and physiological pH [1,2]. Their biological role is not well-understood but likely involves nutrient metabolism, product biosynthesis, hormone degradation, nitrile detoxification, or nutrient assimilation [3]. NHases have attracted substantial interest as biocatalysts in preparative organic chemistry, as they can hydrate a wide range of synthetic nitrile substrates, resulting in their exploitation for biotechnological purposes as biocatalysts in the production of acrylamide and nicotinamide [4]. Industrial, and bioremediation importance of NHase enzymes, a deeper understanding of their reaction mechanism is required to apply and exploit this elegant catalytic chemistry
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