Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

6A, 5, NW12/2004G342

Crystal structure of glucooligosaccharide oxidase from Acremonium strictum
Chun-Hsiang Huang and Liaw, Shwu-Huey
Structural Biology Program, Faculty of Life Science, National Yang-Ming University
Introduction
Sugar oxidases and dehydrogenases that catalyze
carbohydrate oxidation into the corresponding lactones
areofconsiderablecommercialimportance.
Glucooligosaccharideoxidase(GOOX)
fromAcremonium strictum was screened with the aim ofidentifying enzymes with potential applications such as
an oligosaccharide acid production and variousalternative carbohydrate assays. 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 towards oligosaccharides, suggests that it
may have great potential applicability.
To facilitate further characterization and thepotential industrial use of A. strictum GOOX, we havecloned and expressed the encoding gene. Interestingly,
even though GOOX shares some substrate specificity
with glucose oxidase and cellobiose dehydrogenase, it has
no sequence similarity to them. In contrast, GOOX
displays significant sequence homology to the FADbinding domain of plant berberine bridge enzyme-likeproteins, particularly the characteristic flavinylation site(RSGGH). To gain structural insights into the FAD
incorporation, substrate specificity and catalytic
mechanism, we have determined the GOOX structure at
1.55-Å resolution.
Results and Discussion
The protein structure is composed of the FADbinding domain and the substrate-binding domain (Fig. 1).
The F domain consists of two (α + β) subdomains: asmall subdomain, comprising a central four β strands (β1β4), and the second subdomain containing fiveantiparallel β strands (β5-β9). These two subdomains are
surrounded by α-helices, packed against each other, andaccommodate the FAD cofactor between them. The S
domain possesses a large seven-strand antiparallel β sheet(β10-β16) flanked by five helices and constitutes most of
the carbohydrate-binding groove.
Unexpectedly, the FAD cofactor is cross-linked tothe enzyme at two attachment sites. One is the Sγ atom ofCys130 bound to the C6 atom of the isoalloxazine ring,
while the other is the Nδ1 atom of His70 bound to the 8αmethyl group (6-S-cysteinyl, 8α-N1-histidyl FAD). Thisis a novel form of covalent flavinylation; it is the firstexample of 6-S-cysteinyl FAD and is the first double
covalent linkage identified to date.
A variety of carbohydrate molecules were modelled
into the carbohydrate-binding groove using the Insight II
package (Accelrys). The resulting models are consistent

with the substrate specificity from kinetic measurements.
The results suggest that the most significant enzymecarbohydrate contacts are clustered at the +1 and +2
subsites, and weaker interactions extend from the +3 to+4 subsites. D-glucose is the only monosaccharide
substrate for GOOX. Simulation of the complexes showsthat other hexoses and derivatives form either fewerbonds or unfavorable contacts with the amino acidresidues surrounding the substrate-binding groove. The
non-reducing end of the glucose residue can stick out into
the solvent. This explains why, because of the open
carbohydrate-binding groove, the enzyme is able toutilize oligosaccharides as good substrates.
Structure-based mutation analysis regarding theflavinylation, substrate specificity and catalytic
mechanism, are under investigation.

Fig. 1 The GOOX structure. The modeled substratecellobiose (cyan), the cofactor FAD (black) and thelinking residues His70 and Cys130, and the glycosylated
Asn305 and Asn341 are displayed as ball-and-stick
representations. The Four FAD-interacting segments arehighlighted in magenta.
References[1] S. Lin et al., Biochim. Biophy. Acta. 1118, 41 (1991).
[2] W. Lai et al., in submission.
[3] S. Liaw et al., in submission.
* shliaw@ym.edu.tw

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