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. 2005 Dec;71(12):8061-8.
doi: 10.1128/AEM.71.12.8061-8068.2005.

Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in D-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences

Anita Slavica  1 , Iskandar Dib, Bernd Nidetzky
Affiliations

Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in D-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences

Anita Slavica et al. Appl Environ Microbiol. 2005 Dec.

Abstract

One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation in Trigonopsis variabilis d-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.

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Figures

FIG. 1.
FIG. 1.
Separation of different forms of TvDAO. Twenty milligrams of protein was applied on the MonoQ HR 5/5 column and separated (solid line) using seven steps of isocratic flow of 0.1 to 1 M KCl in buffer A (dotted line); TvDAO F1 and F2 were rechromatographed (dashed line) under the same conditions.
FIG. 2.
FIG. 2.
Determination of the N-terminal sequence of TvDAO by nanoLC/ESI-MS/MS. Parent ion at m/z 933.32+ (labeled with a diamond) was analyzed by collision-induced dissociation, and the sequence 4IVVIGAGVAGLTTALQLLR22 of the N-terminal peptide (peptide 2) was deduced.
FIG. 3.
FIG. 3.
ESI tandem mass spectra of the modified peptide 12. (a) ESI-MS/MS spectrum of a precursor ion, [M + 57 + 2H]2+, at m/z 912.42+. The mass of Cys108 has increased by 57 mass units due to carboxamidomethylation. (b) ESI-MS/MS spectrum of a precursor ion, [M + 32 + 2H]2+, at m/z 900.02+. The mass of Cys108 has increased by 32 mass units (two oxygens) in F2.
FIG. 4.
FIG. 4.
Absorption spectra of isolated TvDAO forms. The spectra (F1, solid line; F2, dashed line) were recorded at a protein concentration of 10 μM in 1 mM DTT-10 mM Tris-HCl, pH 7.5. The inset shows the formation of FAD-sulfite complexes of F1 (full circles) and F2 (open circles), expressed as percentages of the total protein concentration, upon the addition of sodium sulfite at 20°C (18, 55, 91, 127, 164, 200, 236, and 273 μM). The quenching of cofactor absorbance at 455 nm served as a reporter of complex formation. The lines (F1, solid line; F2, dashed line) represent sigmoidal fits to the data.
FIG. 5.
FIG. 5.
Kinetic comparison of F1 and F2 in respect to the reduction of dioxygen by enzyme-bound FADH2. Initial rates were recorded by measuring the enzymatic O2 consumption under conditions in which d-methionine was saturating in the steady state (20 mM). The second-order rate constant (kcat/Km) of F1 (1, full circles) and F2 (2, open circles) was obtained from the part of the plot where the enzymatic rate is linearly dependent on the O2 concentration, as indicated in the figure.
FIG. 6.
FIG. 6.
Time courses of aggregation and inactivation of native and oxidatively modified TvDAO under conditions of thermal stress. Activity of 20 μM F1 (open circles) and F2 (open triangles) was monitored at 50°C, and the intensity of scattered light (iS) at 500 nm of F1 (full circles) and F2 (full triangles) at the indicated times was measured. The lines show the trend of the data. AU, arbitrary units.
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