Engineering of nitrilase enantioselectivity for efficient synthesis of brivaracetam
Brivaracetam is a new generation of anti-epileptic drugs with broad market prospects.The nitrilase-catalyzed synthesis of(R)-3-cyanohexanoic acid from 3-cyanocapronitrile,followed by hydrogenation,cyclization and chiral resolution to synthesize brivaracetam is a promising route due to the high atomic economy.However,the poor enantioselectivity of the exploited nitrilases has strongly restricted its application.In this study,the nitrilase PgNIT from Paraburkholderia graminis was exploited.The catalytic pocket and subunit interface domain were modified and a mutant F164W/I202R with significantly improved enantioselectivity was obtained.As a result,the eep value of F164W/I202R was increased from 86％to 97％,while the E value rose from 17 to 111 compared to wild type,respectively.Molecular dynamics simulation analyses revealed that the mutation of F164 reduced the steric hindrance on the entry of(R)-3-cyanocapronitrile into the catalytic pocket.On the other hand,the modification of I202 site at the"A"interface increased the association distance between subunits and increased the dynamic coordination of atoms in the catalytic pocket domain,thus significantly improved the enantioselectivity.
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