FWF - Design einer Nitroaldola - Tuning of a Nitroaldolase by Structure-guided Design

The stereospecific formation of C-C bonds is one of the most important reaction types in preparative organic chemistry. In this respect, biocatalysis is an important and convenient methodology, which generally benefits from excellent stereospecificity, mild reaction conditions and the avoidance of waste problems. The recent development of biocatalytic methods is very promising. However, the total repertoire of reactions types that is available to the (organic) chemist still includes many transformations which have not yet been observed in living systems and for which therefore no suitable enzyme catalyst is presently at hand. One such example is the Henry reaction of nitroaalkanes with aldehydes or ketones to yield ß-nitroalcohols. In this reaction up to two new stereo centres can be created simultaneously and the thus formed nitroalcohols open access to a wide array of follow-up products. We found the hydroxynitrile lyase (HNL) from the tropical rubber tree (Hevea brasiliensis, HbHNL) to exibit nitroaldolase activity and to catalyze the stereospecific addition of nitroalkanes to aldehydes. In vivo, HNLs catalyze the degradation of cyanohydrins. HNLs were not evolved by nature to catalyze stereospecific nitroaldol reactions and are obviously not optimally suited for this task. The project aims at generation of HbHNL variants with largely improved properties for the catalysis of the non-native Henry reaction.

subproject of FWF/ L428 "Struktur-basiertes Design einer Nitroaldolase" (Translational Research Program)

Enzymes are increasingly utilized as catalysts in preparative applications and industrial processes. Thereby one can benefit from the general advantages of biocatalysis - high selectivity, mild reaction conditions, and mostly aqueous media - as well as from ecological advantages, such as lower energy consumption and reduction of dangerous waste. The recent development of biocatalytic methods is very promising. However, the total repertoire of reaction types that is available to the (organic) chemist still includes many transformations which have not yet been observed in living systems and for which therefore no suitable enzyme catalyst is presently at hand. Among those, C-C bond forming reactions are of special importance. One such example is the so called Henry reaction of nitroalkanes with aldehydes or ketones to yield -nitroalcohols. In this reaction up to two new stereo centers can be created simultaneously and the thus formed nitroalcohols open access to a wide array of follow-up products. Very recently, we found the hydroxynitrile lyase (HNL) from the tropical rubber tree (Hevea brasiliensis, HbHNL) to exhibit nitroaldolase activity and to catalyze the stereospecific addition of nitroalkanes to aldehydes. In vivo, HNLs catalyze the degradation of cyanohydrins to release hydrocyanic acid (HCN) and some of these enzymes are used by industry as catalysts for the reverse reaction, i.e. the addition of HCN to carbonyl compounds to obtain enantiopure -hydroxynitriles. However, HNLs were not evolved by nature to catalyze stereospecific nitroaldol reactions and are obviously not optimally suited for this task. Thus, the observed nitroaldolase activities are very low, requiring the addition of about one hundred times more enzyme compared to a corresponding cyanohydrin reaction. Especially this huge amount of catalyst is prohibitive for an industrial application. HbHNL has been studied extensively in our groups with respect to its structure, catalytic mechanism and biocatalytic transformations. Based on our own basic research, the present project aims at generating HbHNL variants with largely improved properties for the catalysis of the non-native Henry reaction. A structure-guided strategy is chosen comprising a limited number of carefully selected, site-directed mutations in the vicinity of the known active site of the enzyme. This approach is highly interdisciplinary and involves structural biology, molecular modeling, molecular biology and organic chemistry. The goal is to boost the nitroaldolase activity of HbHNL to a level at which future applied research could continue developing the enzyme to meet the specific requirements of an industrial process.