Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant

Deacon, Sarah E.; Mahmoud, Khaled; Spooner, R.K.; Firbank, S.J.; Knowles, Peter F.; Phillips, Simon E. V.; McPherson, Michael J.

doi:10.1002/cbic.200300810

Cited by 39 publications

(63 citation statements)

References 26 publications

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“…Even though different mutagenesis techniques were applied to generate M 3-5 , H 1 and F 2 , all three acquired mutations of residue R330. R330A and R330K substitutions were also found to increase the catalytic efficiency of GAO on fructose [101]. Notably, the current comparative analysis of GAO variants engineered for different substrate selectivity and applications underscores the importance of position 330 in substrate recognition, and the potential to identify new applications for previously constructed GAO variants, given the availability of appropriate screens.…”

Section: Altered Substrate Selectivitymentioning

confidence: 90%

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Parikka

Master

Tenkanen

2015

Journal of Molecular Catalysis B: Enzymatic

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Section: Altered Substrate Selectivitymentioning

confidence: 90%

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Parikka

Master

Tenkanen

2015

Journal of Molecular Catalysis B: Enzymatic

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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%

“…The kinetics also showed that D-galactose was a poor substrate for CgRaOx. Notably, the K m value of FgGaOx on D-galactose is also rather high and has been reported at 40 to 100 mM (34,35,41). Moreover, it was surprising that CgRaOx did not oxidize stachyose, given that stachyose specificity rather than galacto-␤-(1¡4)-substituted substrates such as lactose (39,42).…”

Section: Characterization Of a Novel Raffinose Oxidasementioning

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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“…Expression has been increased by using directed evolution and site directed mutagenesis [21]–[23]. Higher yields were obtained by expression of the wild type enzyme in P. pastoris [15], [24]–[27] and A. nidulans [28].…”

Section: Introductionmentioning

confidence: 99%

Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization

et al. 2014

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A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (k cat/Km) was found with 1-methyl-β-D-galactopyranoside (2.2 mM−1 s−1). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.

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Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant

Cited by 39 publications

References 26 publications

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization

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