Naslov
Cys108 is involved in maintenance of D-amino acid oxidase active site environment: a comparison of wild-type enzyme with oxidized, fragmented and mutant variants

Autori
Slavica, Anita ; Nidetzky, Bernd

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
6thEuropean Symposium on Biochemical Engineering Science, Salzburg / Bayer, K. ; Berovič, M. ; Buechs, J. ; Cabral, J.M.S. ; Ferreira, G. N. M. ; Graumann, K. ; Hauer, B. ; Jungbauer, A. ; Luebert, A. ; Mandenius, C.-F. ; Meier, W. ; Nidetzky, B. ; Polakovič, M. ; Posten, C. ; Schlegl, R. ; Schultz, T. ; Thomas, O. - Salzburg : Deutsche Gesellschaft für chemisches Apparatewesen (DECHEMA), 2006, P-280

Skup
6thEuropean Symposium on Biochemical Engineering Science, Salzburg

Mjesto i datum
Salzburg, Austrija, 27.08.2006. - 30.08.2006

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
D-amino acid oxidase; Cys108

Sažetak
Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a FAD-dependent oxidase that catalyzes the oxidative deamination of alpha-D-a, omo acids, coupled to the reduction of the O2 (second substrate) into H2O2. The enzyme is useful biocatalyst that displays absolute D- compared with L-substrate stereoselectivity but relaxed side chain specificity- Immobilized TvDAO is employed in an industrial two-step process for the conversion of cephalosporin C into 7-aminocephalosporanic acid, a well known precursor of semisynthetic beta-lacta, antibiotics. TvDAO is not stable under the process conditions, and the economics of 7-ACA production could benefit significantly from an increase in total turnover number of the oxidase. Three enzyme forms (TvDAO F1, F2, and F3), which differ in structure and activity, were isolated from a technical-grade preparation of TvDAO. F1 is the native enzyme and it undergoes oxidation (Cys108-SH to Cys108-SO2H) as a part of posttranslational modifications in yeast cell or in vitro during application of hypochlorite. The stoichiometric study of the holoenzyme inactivation by sulfhydryl reagents (DTNB, IAA, e.g.) indicates that its complete inactivation is accompanied by the rapid modification of two out from six cysteine residues per subunit. Single-site oxidation (Cys108-SO2H and the e-amino-group of Lys94) and main chain fragmentation (C-terminal of Cys108-SO2H) resulted in weakly active TvDAO form F3. Such unique enzyme form cannot be generated in vitro by chemical oxidation (HOCl) only, indicating that sulfinamide bond formation in protein represents more complex process that sulphur-nitrogen cross-links already reported for synthetic peptides. Mass spectrometry was used to unambiguously assign site specific modification and intramolecular cross-link, both resistant to reduction by dithiothreitol. In the recombinant protein the residue Cys108 was replaced with Asp and Ser by site-directed mutagenesis. Properties of the mutant proteins will be compared with those of the wild-type enzyme.

Izvorni jezik
Engleski

Znanstvena područja
Prehrambena tehnologija

POVEZANOST RADA