Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

Yin, Tyler; Urresti, S.; Lafond, Mickaël; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean‐Guy; Henrissat, Bernard; Walton, Paul H.; Davies, G.J.; Brumer, Harry

doi:10.1038/ncomms10197

Cited by 89 publications

(179 citation statements)

References 71 publications

Supporting

Mentioning

162

Contrasting

Order By: Relevance

“…The glycolaldehyde may not be a natural substrate, since less than 10% of aqueous glycolaldehyde exists in the dimeric ring form, and so the observed activity might be due to the structural resemblance to a sugar ring. Different from the recently discovered alcohol oxidases of the AA5_2 family, CgRaOx showed only very low activity on the tested aliphatic alcohols and thus clearly is a carbohydrate oxidase (30). The sequence identities of CgRaOx with the two alcohol oxidases were only 29 and 30% for CgrAlcOx and CglAlcOx, respectively.…”

Section: Characterization Of a Novel Raffinose Oxidasecontrasting

confidence: 59%

“…Applied and Environmental Microbiology discovery of Colletotrichum graminicola and C. gloeosporioides alcohol oxidases (CgrAlcOx and CglAlcOx), which also belong to the AA5_2 subfamily (30). The sequence of CgRaOx characterized here is a AA5_2 carbohydrate oxidase with a distinct substrate profile.…”

Section: Characterization Of a Novel Raffinose Oxidasementioning

confidence: 84%

“…The three other carbohydrate oxidase candidates were also identified as AA5_2 members and, interestingly, the gene for G4NG45 showed high sequence identity (ca. 57%) with the two recently discovered novel alcohol oxidases (AlcOx) (30). The full-length sequence of CgRaOx is 913 amino acids (aa), and the predicted native signal sequence is 23 residues long.…”

Section: Resultsmentioning

confidence: 99%

“…The corresponding residue in FgGaOx, i.e., Arg330, has been reported to form hydrogen bonds to the C-3 and C-4 hydroxyls of galactose and thus, together with the copper-ligand Tyr495, is the main explanation for the exclusion of glucose as a substrate. In Cgr AlcOx the Trp290 is replaced by a phenylalanine, which Yin et al suggest enhances the ability of the enzyme to oxidize alcohols (30). However, CgRaOx, with a tyrosine at Trp290, clearly showed greater activity on sugars than on alcohols.…”

Section: Characterization Of a Novel Raffinose Oxidasementioning

confidence: 99%

“…However, K m values of CgRaOx on glycolaldehyde (K m ϭ 185 mM) were lower than those measured using raffinose (apparent K m ϭ 480 mM). Two recently published alcohol oxidases from AA5_2 showed activity toward a range of simple primary alcohols but not on sugars (30). Therefore, CgRaOx was also tested on a range of simple alcohols (Fig.…”

Section: Andberg Et Al Applied and Environmental Microbiologymentioning

confidence: 99%

See 4 more Smart Citations

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Andberg

Mollerup

Parikka

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

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

show abstract

Section: Characterization Of a Novel Raffinose Oxidasecontrasting

confidence: 59%

Section: Characterization Of a Novel Raffinose Oxidasementioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Characterization Of a Novel Raffinose Oxidasementioning

confidence: 99%

Section: Andberg Et Al Applied and Environmental Microbiologymentioning

confidence: 99%

See 3 more Smart Citations

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Andberg

Mollerup

Parikka

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

A unique AA5 alcohol oxidase fused with a catalytically inactive CE3 domain from the bacterium Burkholderia pseudomallei

2023

View full text Add to dashboard Cite

Copper radical oxidases (CROs) are redox enzymes able to oxidize alcohols or aldehydes, while only requiring a single copper atom as cofactor. Studied CROs are found in one of two subfamilies within the Auxiliary Activities family 5 (AA5) in the carbohydrate-active enzymes database. We here characterize an AA5 enzyme outside the subfamily classification from the opportunistic bacterial pathogen Burkholderia pseudomallei, which curiously was fused to a carbohydrate esterase family 3 domain. The enzyme was shown to be a promiscuous primary alcohol oxidase, with an activity profile similar to enzymes from subfamily 2. The esterase domain was inactive on all tested substrates, and structural predictions revealed this being an effect of crippling substitutions in the expected active site residues.

show abstract

Radical Enzymes

Högbom

Sjöberg

Berggren

2020

Encyclopedia of Life Sciences

View full text Add to dashboard Cite

Radical enzymes harbour a free radical in their polypeptide chain, which participates in catalysis. A growing number of enzymes are known to require a posttranslationally generated free radical for their proper functioning. The radical is generally used to remove a hydrogen atom from an unreactive position in the substrate, activating the substrate to undergo difficult chemistry. Some radical enzymes have unexpectedly been found to harbour a stable radical on an amino acid side‐chain that is distinct from the side‐chains of the active site region; during catalysis, the stable free radical interacts with the active site via radical transfer, and between turnovers it serves as a radical sink. Key Concepts Radical enzymes utilise single‐electron chemistry to enable difficult chemical reactions. Tyrosyl and tryptophanyl radicals are involved in a large number of biochemical reactions as initiators of catalysis or as partners in radical transfer pathways. Glycyl radicals are oxygen‐sensitive and have only been found in anaerobically expressed enzymes. Transient cysteinyl radicals are important intermediates in a number of radical enzymes. A transient 5′‐deoxyadensyl radical can be formed either by reductive cleavage of the S‐ adenosylmethionine cofactor in a radical SAM enzyme or by homolytic cleavage of the vitamin B12 coenzyme AdoCbl. All members of the ribonucleotide reductase enzyme family utilise a transient cysteinyl radical generated by a stable radical in a separate subunit (class I), cleavage of AdoCbl (class II), or a stable glycyl radical (class III).

show abstract

Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

Cited by 89 publications

References 71 publications

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

A unique AA5 alcohol oxidase fused with a catalytically inactive CE3 domain from the bacterium Burkholderia pseudomallei

Radical Enzymes

Contact Info

Product

Resources

About