Pyranosone dehydratase from the basidiomycete Phanerochaete chrysosporium: improved purification, and identification of 6-deoxy-D-glucosone and D-xylosone reaction products.
Pyranose oxidase and pyranosone dehydratase (aldos-2-ulose dehydratase), enzymes which convert in coupled reactions D-glucose to beta-pyrone cortalcerone, peaked coincidently during idiophasic growth of Phanerochaete chrysosporium under agitated conditions. The enzymes were purified from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phenyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD II, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange chromatography on Mono Q to yield apparently pure enzyme as judged by SDS-PAGE and gel filtration on Superose 12. Isoelectric focusing indicated microheterogeneity of the protein by the presence of at least five protein bands with pI 5.1-5.3. PD II had a pI of 5.75. Overall PD I purification was 60.7-fold with 50% yield. The enzyme acted on several osones (glycosuloses), with the preferred substrate being D-glucosone. D-Xylosone and 6-deoxy-D-glucosone were dehydrated at C-3-C-4 to give the corresponding 5-hydroxy-2,3-dioxoalcanals (4-deoxy-2,3-glycosdiuloses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine derivatives and spectroscopically identified. The analogous D-glucosone dehydration product did not accumulate due to its further transformation. pH optimum of PD I activity was 6.0 and its pH stability was optimal at pH 7-11. The enzyme was sensitive to Me2+ chelating agents and some heavy metal ions (Hg2+, Cu2+).
Gabriel
J
J
Institute of Microbiology, Academy of Sciences of Czech Republic, Prague.
Volc
J
J
Sedmera
P
P
Daniel
G
G
Kubátová
E
E
D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis; D002614:Chelating Agents / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007661:Ketoses / Q000096:biosynthesis / Q000378:metabolism*; D013379:Substrate Specificity
D002614:Chelating Agents; D007661:Ketoses; C067906:threo-pentos-2-ulose; C082558:6-deoxyglucosone; D003847:Deoxyglucose; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D006836:Hydro-Lyases; C082557:pyranosone dehydratase