Amino acids are among the most important molecules in nature and chemistry since they play central roles both as intermediates in metabolism and as building blocks of proteins and pharmaceuticals. In living organisms enzymes that depend on pyridoxal-5’-phosphate (PLP) catalyze a wide variety of biochemical reactions involving amino acids substrates and their analogs. These enzymes have been exploited by chemists for the biocatalytic synthesis of natural amino acids, and most recently have been investigated for the asymmetric synthesis of non-canonical amino acids. During the project we will explore and broaden the catalytic potential of PLP-dependent lyases, in particular threonine aldolases, for the application as biocatalysts to produce alpha- and beta-substituted alpha-amino acids. The high specificity towards natural substrates and sometimes low stereoselectivity is the limiting factor for the use of the PLP-dependent lyases in organic synthesis. Rational design represents an efficient approach for engineering of enzymes with desired properties. However, identification of functional residues is challenging, especially when structure-function relationships in an enzyme is poorly understood. Looking back to the evolution of enzymes within one family of PLP-dependent enzymes is essential to understand how functionalities were evolved by Nature. The evolution based knowledge will be used to design or evolve new substrate specificities and catalytic activities in the enzymes of interest. During the research on PLP-dependent enzymes, their genetic and structural information will be compared using modern bioinformatic tools in order to:
a) increase knowledge on structure-function relationships in PLP-dependent enzymes and identify the amino acid residues responsible for functional discrimination between enzymes sharing common evolutionary origin;
b) predict mutations necessary for improving substrate (e.g. donor) specificity and diastereoselectivity of threonine aldolases and design catalysts for the synthesis of beta-hydroxy alpha-quaternary-alpha-amino acids;
c) expose the catalytic potential of PLP-dependent lyases with versatile catalytic properties (e.g. tryptophan synthase, tyrosine phenol-lyase and cystathionine beta-lyase) and evolve their promiscuous activities for the formation of beta-substituted alpha-amino acids;
Rationally designed and evolved lyases will be applied for the asymmetric synthesis of non-canonical amino acids using a novel biocatalytic approach with high selectivity under mild reaction conditions, which will be a promising alternative towards currently used organic chemistry methods.