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GtHNL from Granulicella tundricola is a Mn(II) containing hydroxynitrile lyase with a cupin fold. The quasi-octahedral manganese is pentacoordinated by the enzyme. It catalyzes the enantioselective addition of HCN to aldehydes, yielding R-cyanohydrins. On the Lewis acidic vacant coordination site the Mn binds either substrate or the product, leading to a hexacoordinated 17 electron complex. EPR spectra of the active enzyme are unusually wide with a zero-field splitting approximately equal to the X-band microwave energy. A spectral change is induced by incubation with either one of the substrates/products HCN, benzaldehyde, and/or mandelonitrile. This points toward Mn(II) catalyzed cyanohydrin synthesis.

Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope.