Switching a nitrilase from Syechocystis sp. PCC6803 to a nitrile hydratase by rationally regulating reaction pathways

Abstract

Based on this mechanism, a nitrilase was engineered to shift the reaction pathway from formation of acid to formation of amide.

Inverting the Enantiopreference of Nitrilase‐Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror‐Image Strategy

“…Variation in amide 5 a formation was studied by MD sampling of the wt Ss NIT and three mutants. The results showed that NH 2 group in the tetrahedral intermediate for the mutant was more easily protonated and resulted in less amide 5 a formation, being consistent with the studies by Jiang et al [24] . (Supporting Information, Table S10).…”

Interplay of nucleophilic catalysis with proton transfer in the nitrile reductase QueF from Escherichia coli

“…QueF and nitrilase appear to utilize a highly similar mechanism to promote the covalent thioimidate intermediate (Scheme 1A). 9,14,41 In the nitrilase, a triad of cysteine/ glutamic acid/lysine constitutes the active-site apparatus. 8,16,41 Like Asp197 in QueF, the glutamic acid is central for catalytic nucleophile activation and for acid-base catalysis.…”

“…[1][2][3] They also have significant applications in synthetic chemistry wherein nitriles often represent important intermediates. 4,5 The nitrile group is converted by hydrolytic enzymes to an amide or to a carboxylic acid [6][7][8][9] and by reductive enzymes to an amine. [10][11][12][13] Despite the different reactivities, nitrile-converting enzymes share covalent catalysis from an active-site nucleophile as a common feature of their mechanisms.…”

“…[10][11][12][13] Despite the different reactivities, nitrile-converting enzymes share covalent catalysis from an active-site nucleophile as a common feature of their mechanisms. Nitrilase 8,9,[14][15][16] and nitrile reductase [17][18][19][20] both utilize a cysteine to form a thioimidate adduct between the enzyme and nitrile substrate (Scheme 1A). The covalent catalysis in each enzyme requires assistance from catalytic proton transfer.…”

“…Conversion of nitrile to the covalent intermediate and further on to the product necessitates acid-base catalysis, as shown in Scheme 1A. Nitrile reductase and nitrilase both have a candidate acid-base residue (Asp or Glu) positioned close to their cysteine nucleophile 8,9,11,16,18,21 but the precise role of the Asp/Glu remains to be elucidated. Moreover, the critical interplay between nucleophilic and general acid-base catalysis is not well understood in both enzymes.…”