Crystal Structure of Glucooligosaccharide Oxidase from Acremonium strictum

Abstract

Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello-and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxycellobiono-1,5-lactam at 1.55-and 1.98-Å resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8␣-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C Sugar oxidases and dehydrogenases that catalyze carbohydrate oxidation to the corresponding lactones are of considerable commercial importance as potential diagnostic reagents and industrial biocatalysts. For example, glucose oxidase (GOX) 4 is widely used in analytical biochemistry and in the food industry (1). A search of the BRENDA enzyme data base (2) reveals that most of these enzymes are specific for a variety of mono-and disaccharides, and only a few enzymes are highly selective for oligosaccharides. These include galactose oxidase, cellobiose dehydrogenase (CDH), and glucooligosaccharide oxidase (GOOX). GOOX from Acremonium strictum was screened with the aim of identifying enzymes with potential applications in oligosaccharide acid production and alternative carbohydrate assays (3). It is a monomeric glycoprotein with a covalently linked FAD and catalyzes the oxidization of a variety of carbohydrates with the concomitant reduction of molecular oxygen to hydrogen peroxide. A screening of more than 50 carbohydrates and derivatives showed that D-glucose, maltose, lactose, and cellobiose are good substrates. In addition, this enzyme can react with malto-and cellooligosaccharides, and hence the name of this novel oxidase. The broad substrate specificity of GOOX, particularly toward oligosaccharides, suggests that it may have great potential applicability.To facilitate further characterization and potential industrial use of A. strictum GOOX, we have cloned the encoding gene, which is composed of a 25-residue signal peptide and a 474-residue mature protein (4). Although GOOX shows a similar substrate specificity as GOX, CDH, and pyranose oxidase (POX), it shares no sequence similarity with them. However, GOOX displays significant sequence homology to the FADbinding domain (F domain) of berberine bridge enzyme-like proteins including three other sugar oxidases, a red alga hexose oxidase (CcHEOX), a tobacco nectar protein (nectarin V, NlNEC5), and a sunflower defense protein (HaCHOX) (5-7). Interestingly, although NlNEC5 catalyzes oxidation of glucose, and GOOX, CcHEOX and HaCHOX catalyze the oxidation of glucose, maltose, lactose, and cellobiose, the protein sequences of their substrate-binding domains (S domains) are quite diverse and apparently lack conserved carbohydrateinteracting residues. Moreover, structural and mutational studies demonstrated a consensus histidine for f...

A concerted mechanism for berberine bridge enzyme

“…1c and Supplementary Fig. 2 online), thus providing an additional example for the recently discovered group of bi-covalently flavinylated enzymes [6][7][8][9][10] . The active site cavity close to the cofactor is lined by mainly hydrophobic residues with the only notable exceptions being Tyr106, Thr358, Asn390, Glu417 and His459 (Fig.…”

6-S-Cysteinylation of Bi-covalently Attached FAD in Berberine Bridge Enzyme Tunes the Redox Potential for Optimal Activity

“…Recently, we have developed an expression system in Pichia pastoris that enabled us to obtain sufficient quantities of purified BBE from Eschscholzia californica, allowing a more detailed characterization. In the course of these studies it was discovered that BBE belongs to a novel group of flavoproteins containing a bi-covalently attached flavin cofactor (3)(4)(5)(6)(7).…”

“…Several explanations for the occurrence of covalent flavinylation have been given for different enzymes, ranging from prevention of cofactor modification in the case of trimethylamine dehydrogenase (4,9) or increasing the redox potential of flavins modified in position 8␣ (10,11) to facilitating electron transfer from the flavin to other redox centers present in p-cresol methylhydroxylase (12). In addition to this variety of explanations for the existence of a covalent linkage, the situation is further complicated by the occurrence of covalent flavoenzymes having isoenzymes bearing a dissociable cofactor but still exhibiting a similar kinetic behavior (13,14).…”

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

“…About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15,16), hexose oxidase (17), and berberine bridge enzyme (18,19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20 -22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20,(23)(24)(25)(26)(27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20).…”