Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution

Rannes, Julie Bille; Ioannou, Avgousta; Willies, Simon C.; Grogan, Gideon; Behrens, Carsten; Flitsch, Sabine L.; Turner, Nicholas J.

doi:10.1021/ja2018477

Cited by 106 publications

(128 citation statements)

References 31 publications

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“…From this collection, those that lacked residues predicted to act as copper ligands or else lacked the cysteine involved in the Tyr-Cys thioether bond were excluded from the set. Previous protein engineering and docking studies of FgGaOx identified Arg330, Phe464, Gln406, Phe194, Trp290, Tyr405, and Pro463 as potentially important for substrate binding (24,33,34). Therefore, sequences that differed at one or more of these positions, relative to the FgGaOx residues, were selected.…”

Section: Resultsmentioning

confidence: 99%

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Andberg

Mollerup

Parikka

et al. 2017

Appl Environ Microbiol

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We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.Ϫ) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on D-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1 H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O 2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.KEYWORDS CAZy AA5, galactose oxidase, nuclear magnetic resonance, NMR, carbohydrate, EC 1.1.3.Ϫ E nzymes that cleave or form oligo-and polysaccharides are classified in the sequence-based carbohydrate-active enzyme database (CAZy [http://www.cazy .org]). The sequences are linked to information about functionality (e.g., substrate specificity) and three-dimensional structure. The CAZy database was recently expanded to account for redox enzymes, leading to the auxiliary activities (AA) group. Currently, 13 AA families exist, comprising lignolytic oxidoreductases (families AA1, -2, -4, and -6), lytic polysaccharide monooxygenases (families AA9, -10, -11, and -13), carbohydrate

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Section: Resultsmentioning

confidence: 99%

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Andberg

Mollerup

Parikka

et al. 2017

Appl Environ Microbiol

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“…In addition to constructing a glucose 6-oxidase from GAO, enzyme engineering efforts have created GAO variant (M [3][4][5] ) that catalyse the enantioselective oxidization of secondary alcohols [98] and GAO variants (H 1 and F 2 ) that selectively oxidize glycans on glycoproteins [99]. While variant M [3][4][5] has been used to synthesize optically pure secondary alcohols and atropisomeric diaryl ethers [98,100], improved activity of H 1 and F 2 on D-mannose and D-N-acetyl glucosamine enabled selective labelling of glycoproteins in solution or present on the surface of whole cells [99].…”

Section: Altered Substrate Selectivitymentioning

confidence: 99%

“…While variant M [3][4][5] has been used to synthesize optically pure secondary alcohols and atropisomeric diaryl ethers [98,100], improved activity of H 1 and F 2 on D-mannose and D-N-acetyl glucosamine enabled selective labelling of glycoproteins in solution or present on the surface of whole cells [99]. Even though different mutagenesis techniques were applied to generate M 3-5 , H 1 and F 2 , all three acquired mutations of residue R330.…”

Section: Altered Substrate Selectivitymentioning

confidence: 99%

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Parikka

Master

Tenkanen

2015

Journal of Molecular Catalysis B: Enzymatic

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“…169 Toward expanding the substrate scope of galactose oxidase, directed evolution was used to select for enzyme variants that could oxidize mannose and GlcNAc residues. 172 Recently, Kohler and co-workers utilized both enzymatic and chemical oxidation to enrich and identify mammalian glycoprotein substrates for bacterial sialidases. 173 …”

Section: Enrichment Of Glycoproteins and Glycopeptidesmentioning

confidence: 99%

Chemical Glycoproteomics

Palaniappan

Bertozzi²

2016

Chem. Rev.

235

205

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Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

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Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution

Cited by 106 publications

References 31 publications

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Chemical Glycoproteomics

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