Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant

Mirgorodskaya, Ekaterina; Fierobe, Henri‐Pierre; Svensson, Birte; Roepstorff, Peter

doi:10.1002/(sici)1096-9888(199909)34:9<952::aid-jms856>3.0.co;2-y

J. Mass Spectrom.

1999

DOI: 10.1002/(sici)1096-9888(199909)34:9<952::aid-jms856>3.0.co;2-y

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Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant

Ekaterina Mirgorodskaya¹,

Henri‐Pierre Fierobe

Birte Svensson

et al.

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Cited by 3 publications

(1 citation statement)

References 22 publications

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“…Bacterial tetrachlorohydrochinone dehalogenase is inactivated through the oxidation of its Cys13 to a sulfenic acid (36). A catalytic mutant of glucoamylase, Glu4003 Cys, could be reactivated by oxidation of the cysteine to a sulfinic acid (18). In the physiological context, nitrile hydratase undergoes posttranslational oxidation at two active-site cysteines, thereby generating a unique Cys-SOH-and Cys-SO 2 H-containing catalytic center in the fully active enzyme (19).…”

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confidence: 99%

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

Slavica

Dib

Nidetzky

2005

Appl Environ Microbiol

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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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mentioning

confidence: 99%

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

Slavica

Dib

Nidetzky

2005

Appl Environ Microbiol

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Glucoamylase: structure/function relationships, and protein engineering

Sauer

Sigurskjold

Christensen

et al. 2000

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

186

120

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Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry

Larsen

Højrup

Roepstorff

2005

Molecular & Cellular Proteomics

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Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-ofthe-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies. Glycosylation is one of the most abundant post-translational protein modifications in nature. The biological role of glycosylation varies from conformational stability and protection against degradation to molecular and cellular recognition in development, growth, and cellular communication (1, 2). Several diseases have been associated with abnormalities in carbohydrate degradation and recognition (3, 4). The majority of glycosylated proteins are secreted or membrane-bound. Many recombinant proteins produced by the biotechnology industry are glycoproteins, e.g. cytokines or antibodies. These require a correct glycan structure for optimal biological function and to avoid triggering an immune response. Therefore, faster and more sensitive methods for characterizing glycoproteins are particularly important to understand their biological functions and to verify the structure of recombinant glycoproteins.Four types of glycosylation are known: N-linked where the sugar is attached to asparagine residues in the consensus sequence Asn-Xaa-(Ser/Thr/Cys); O-linked where the sugar is attached to serine or threonine; glycosylphosphatidylinositol anchors, which are attached to the carboxyl terminus of certain membrane-associated proteins; and finally C-glycosylation, which has been found attached to tryptophan residues in certain membrane-associated and secreted proteins (5). Except for the latter two glycosylation types, each glycosylated amino acid may have a vide variety of different glycan structures attached, leading to a pronounced heterogeneity (microheterogeneity). The consequence is that for a given amount of protein there is a significantly lower amount of each glycosylated isopeptide compared with the non-modified peptide from a glycosylated protein. In addition, different sites may be only partially glycosylated (macroheterogeneity), resulting in more complex mixtures and ambiguous glycosylation site assignment. Mass spectrometric analysis of glycopeptides is further ...

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Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant

Cited by 3 publications

References 22 publications

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

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

Glucoamylase: structure/function relationships, and protein engineering

Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry

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