Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.
Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.
Scientific Reports 7: Article number: 46738; published online: 03 May 2017; updated: 26 May 2017 The original version of this Article contained errors. In the Introduction section, “Finally there is a number of characterized HNLs with yet unpublished amino acid sequences and protein folds, for example, PatHNL (Prunus amygdalus turcomanica)25, ParsHNL (Prunus armeniaca L.
Background The accumulation of intracellular fat depots is a polygenic trait. Therefore, the extent of lipid storage in the individuals of a species covers a broad range and is determined by many genetic factors. Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. We used this method and complementary approaches to identify genes in the yeast Saccharomyces cerevisiae that are involved in neutral lipid storage. Results We selected two yeast strains, the laboratory strain BY4741 and the wine yeast AWRI1631, with a more than two-fold difference in neutral lipid content. After crossing, sporulation and germination, we used fluorescence activated cell sorting to isolate a subpopulation of cells with the highest neutral lipid content from the pool of segregants. Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. Three of the identified genes, PIG1, PHO23 and RML2, were investigated in more detail. Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. cerevisiae and that PIG1, PHO23 and RML2 are the major causative genes. Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype. Conclusions We identified several genes that contribute to the phenotype of lipid accumulation in an allele-specific manner. Surprisingly, no allelic variations of genes with known functions in lipid metabolism were found, indicating that the level of storage lipid accumulation is determined by many cellular processes that are not directly related to lipid metabolism.
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