Engineering of pyranose dehydrogenase for increased oxygen reactivity

• Published in: PloS one 2014-03, Vol.9 (3), p.e91145
• Main Authors: Krondorfer, Iris, Lipp, Katharina, Brugger, Dagmar, Staudigl, Petra, Sygmund, Christoph, Haltrich, Dietmar, Peterbauer, Clemens K
• Format: Article
• Language: English
• Subjects: Agaricus - enzymology; Amino acids; Biology; Biotechnology; Carbohydrate Dehydrogenases - metabolism; Catalytic Domain; Chemical Precipitation; Dehydrogenase; Dehydrogenases; Dextrose; E coli; Electrons; Electrophoresis, Polyacrylamide Gel; Enzymes; Flavin-adenine dinucleotide; Flavoproteins; Food processing; Genes; Genetic Engineering - methods; Glucose; High-throughput screening; High-Throughput Screening Assays; Histidine; Horseradish peroxidase; Kinetics; Laboratories; Life sciences; Metals; Methionine; Mutagenesis; Mutagenesis, Site-Directed; Mutant Proteins - metabolism; Natural resources; Oxygen; Oxygen - pharmacology; Peroxidase; Phenylalanine; Pichia pastoris; Plasmids; Potassium; Quinones; Reactivity; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - metabolism; Saturation mutagenesis; Substitutes; Substrate specificity; Substrates; Temperature; Tryptophan; Tyrosine; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0091145

Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.