Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

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.

Trigonopsis variabilisd-Amino Acid Oxidase: Control of Protein Quality and Opportunities for Biocatalysis through Production inEscherichia coli

“…The specific activity of TvDAOstrepN was 160 U/mg, as assayed by measurement of enzymatic oxygen consumption rates with a fiber optic microsensor (with D-Met as the substrate [18]). Native oxidase isolated from T. variabilis (16) had the same specific activity. Exogenous flavin adenine dinucleotide (FAD) (10 to 1,000 M) added to assay or protein storage buffers did not enhance the catalytic activity of native and recombinant TvDAO.…”

“…The technical-grade oxidase obtained in this way is structurally microheterogeneous due to the partial oxidation of Cys108 into a cysteine sulfinic acid (16). Oxidatively modified TvDAO is catalytically only about one-quarter as efficient as the native enzyme (16). Production of a recombinant TvDAO potentially could eliminate many shortcomings of the established process, especially in a prokaryotic host with a reducing cellular environment that should prevent cysteine oxidation in TvDAO.…”

“…Current industrial enzyme production integrates relatively slow, multistage bioreactor cultivation of T. variabilis and several steps of downstream processing to obtain an immobilized biocatalyst deficient in catalase and esterase activities (8,14). The technical-grade oxidase obtained in this way is structurally microheterogeneous due to the partial oxidation of Cys108 into a cysteine sulfinic acid (16). Oxidatively modified TvDAO is catalytically only about one-quarter as efficient as the native enzyme (16).…”

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

“…For example, H 2 O 2 can irreversibly inactivate the human brain type of creatine kinase via a two-stage process, via modification of CYS283, the catalytic residue [91]. Tyrosine phosphatases [92], porcine kidney betaine aldehyde dehydrogenase [93], and D-amino acid oxidase [90,94] are inactivated in a similar way.…”

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

“…One of the most common modifications is oxidation of labile amino acid side chains, which is induced by reactive oxygen species. 1 Although protein oxidation can occur at cysteine, tryptophan, lysine and other amino acids, [2][3][4] methionine is often the most susceptible residue to oxidation. 5,6 The most common product of methionine oxidation is methionine sulfoxide, which is more polar and less hydrophobic than methionine.…”