TAILIEUCHUNG - Báo cáo khoa học: Characterization of the flavin association in hexose oxidase from Chondrus crispus

Hexose oxidase (EC ) fromHansenula polymorphawas found to exhibit a dual covalent association of FAD with His79 via an 8a-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD. Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. | ềFEBS Journal Characterization of the flavin association in hexose oxidase from Chondrus crispus Thomas Rand1 Karsten B. Qvist1 Clive P. Walter1 and Charlotte H. Poulsen1 2 1 Danisco A S Brabrand Denmark 2 Interdisciplinary Nanoscience Center iNANO University of Aarhus Denmark Keywords FAD flavin hexose oxidase mass spectroscopy modeling Correspondence T. Rand Danisco A S Edwin Rahrs Vej38 8220 Brabrand Denmark Fax 45 8625 1077 Tel 45 8943 5000 E-mail Received 20 January 2006 revised 17 April 2006 accepted 20 April 2006 doi Hexose oxidase EC from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8a-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD. Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. An H79K mutant enzyme exhibited only one maximum at 437 nm. The difference absorption spectrum between an oxidized and a substrate-reduced enzyme preparation showed maxima at 360 nm and 445 nm corresponding to an apparent novel type of association. Hexose oxidase showed a low pH-independent fluorescence at 525 nm when excited at 450 nm. Flavin was released from the holoenzyme by treatment with trypsin. Sequencing of the flavopeptide revealed two peptides comprising positions 74-91 and 132-157 associated with FAD in equimolar amounts. A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template. The model placed both of the sequences found above in the close vicinity of the FAD cofactor and suggests covalent bonds between both His79 and Cys138 and FAD in accordance with the chemical evidence. Based on the results hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holoenzyme. A reaction .

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