ИСТИНА

Background: D-amino acid oxidase (DAAO) plays very important role in prokaryotes and eukaryotes. Also it widely used in different industrial fields (including preparation of 7-aminocephalosporanic acid), fine organic synthesis, analytical biotechnology and medical diagnostics. DAAOs from different sources (particularly in bacteria) show very low aa sequence homology (in many cases less than 25%) and this approach does not permit to select DAAO with required properties. Objective: To develop and test approach to search in genomes genes of new DAAOs with desired properties. Methods: Bioinformatic approach was applied to find tentative DAAO genes in genomes of thermotolerant yeast Ogataea parapolymorpha and extremophile microorganisms. Genetic engineering and biochemical methods were used to obtain and characterize new Opa and bacterial DAAOs. Structure experiments included 3D modeling with AlphaFold and crystallization and X-ray analysis. Results: We found 6 ORFs of possible DAAOs in genome O. parapolymorpha. Five genes were cloned and expressed in E.coli. All enzymes had oxidase activity and 3 DAAOs were unique without analogs in literature. 3D model structures of all OpaDAAOs prepared using AlphaFold approach. Two OpaDAAOs were crystallized and RSA structures were solved with resolution 1.8 Å. 3D model and RSA structures were very close (RSMD 0.5 Å or less) and positions of aa residues in substrate-binding domain nicely explained substrate specificity. FASTA search followed by 3D structure modeling and analysis were used to find new DAOO genes in >1 000 genomes of extremophile bacteria. Two new DAAOs specific to aromatic aa were selected. The genes were cloned and expressed in E.coli. Enzymes showed high stability and expected catalytic properties. Conclusion: Combination of sequence homology search followed by 3D structure modeling is very efficient and powerful approach to look for new DAAO genes with very low sequence homology. New unique bacterial recombinant DAAOs were found and prepared. This work was supported in part by Russian Ministry of Science and Higher Education (Agreement 075-15-2021-1396).