Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access. 2018

Kalyanasundaram Subramanian, and Artur Góra, and Ruud Spruijt, and Karolina Mitusińska, and Maria Suarez-Diez, and Vitor Martins Dos Santos, and Peter J Schaap
Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Stippeneng WE, Wageningen, The Netherlands.

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

UI MeSH Term Description Entries
D003605 D-Amino-Acid Oxidase dextro-Amino Acid Oxidase,D-Amino Acid Dehydrogenase,Acid Dehydrogenase, D-Amino,Acid Oxidase, dextro-Amino,D Amino Acid Dehydrogenase,D Amino Acid Oxidase,Dehydrogenase, D-Amino Acid,Oxidase, D-Amino-Acid,Oxidase, dextro-Amino Acid,dextro Amino Acid Oxidase
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D001709 Biotechnology Body of knowledge related to the use of organisms, cells or cell-derived constituents for the purpose of developing products which are technically, scientifically and clinically useful. Alteration of biologic function at the molecular level (i.e., GENETIC ENGINEERING) is a central focus; laboratory methods used include TRANSFECTION and CLONING technologies, sequence and structure analysis algorithms, computer databases, and gene and protein structure function analysis and prediction. Biotechnologies
D012997 Solvents Liquids that dissolve other substances (solutes), generally solids, without any change in chemical composition, as, water containing sugar. (Grant & Hackh's Chemical Dictionary, 5th ed) Solvent
D013379 Substrate Specificity A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D057075 Enzyme Assays Methods used to measure the relative activity of a specific enzyme or its concentration in solution. Typically an enzyme substrate is added to a buffer solution containing enzyme and the rate of conversion of substrate to product is measured under controlled conditions. Many classical enzymatic assay methods involve the use of synthetic colorimetric substrates and measuring the reaction rates using a spectrophotometer. Enzymatic Assays,Indirect Enzymatic Assays,Indirect Enzyme Assays,Assay, Enzymatic,Assay, Enzyme,Assay, Indirect Enzymatic,Assay, Indirect Enzyme,Assays, Enzymatic,Assays, Enzyme,Assays, Indirect Enzymatic,Assays, Indirect Enzyme,Enzymatic Assay,Enzymatic Assay, Indirect,Enzymatic Assays, Indirect,Enzyme Assay,Enzyme Assay, Indirect,Enzyme Assays, Indirect,Indirect Enzymatic Assay,Indirect Enzyme Assay

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