24 Crystal Structure of Glucooligosaccharide Oxidase f r o m Acremonium strictum – A Novel Flavinylation o f 6 - S -cysteinyl, 8 α - N1-histidyl FAD Glucooligosaccharide oxidase (GOOX) 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 malto-oligosaccharides (Fig. 1) [1]. Here we have determined the GOOX structure in the absence, or presence of a product analog, 5- amino-5-deoxy-cellobiono-1,5-lactam (ABL) ( BL12B2 ) (Fig. 2(a)) [2]. Unexpectedly, the FAD cofactor is cross-linked to the enzyme at two attachment sites (Fig. 2(b)). One is the S γ atom of Cys 130 bound to the C 6 atom of the isoalloxazine ring, while the other is the N δ 1 atom of His 70 bound to the 8 α -methyl group (6- S -cysteinyl, 8 α - N 1-histidyl FAD). Five types of covalent flavinylation have been identified up to the present and flavinylation has been shown to be an autocatalytic process [3]. The four types of flavinylation at the 8 α -methyl group cross-link to His (N δ 1 and N ε 2 ), Tyr (O η ) and Cys (S γ ), whereas an unusual 6-S-cysteinyl modification has been observed only in the FMN cofactor of trimethylamine dehydrogenase and its homologues. Thus, GOOX possesses a novel form of covalent flavinylation; it is the first example of 6- S -cysteinyl FAD and the first double covalent linkage identified to date. The His 70 and Cys 130 mutants suggest that the covalent attachment is able to enhance the redox potential of the flavin [1]. ABL is firmly embedded on the si face of the isoalloxazine ring without induction of any significant conformational change except for Glu 247 (Fig. 3(a)). A variety of carbohydrate molecules were then modeled manually in the substrate-binding groove. D-glucose is the only monosaccharide substrate for GOOX. Simulation of the complexes shows that other hexoses and derivatives form either fewer hydrogen bonds or unfavorable contacts with the surrounding residues. GOOX possesses an open carbohydrate-binding groove, which explains why the enzyme is able to utilize oligosaccharides as good substrates (Fig. 3(b)). The substrate cellobiose was modeled into the active site, suggesting that GOOX preferentially oxidizes the β anomer with the conserved Tyr 429 acting as a general base. As is common for flavoenzymes, the reaction mechanism of GOOX consists of two half- reactions (Fig. 1). The reductive half-reaction is involved in the oxidation of the free reducing-end β -D- glucosyl residue to glucono-1,5-lactone by hydride transfer to the N 5 atom, probably initiated by proton abstraction from the OH 1 group by Tyr 429 . The lactone product is spontaneously hydrolyzed to gluconic acid. In the oxidative half-reaction, regeneration of the oxidized FAD by molecular oxygen yields hydrogen peroxide. According to the structural fold, flavoenzymes have been classified into many superfamilies [4]. Interestingly, folding topology does not correlate with enzyme function. For example, GOOX and Brevibacterium sterolicum cholesterol oxidase 2 (BsCOX2) belong to the p -cresol methylhydroxylase (PCMH) superfamily, whereas glucose oxidase, Fig. 1. The proposed catalytic mechanism for GOOX. S N N N NH O O C H 3 H 2 C N N R H O H OH O OH O CH 2 OH O H O O O H H H S N N H N NH O O C H 3 H 2 C N N R OH OH OH O CH 2 OH O H O OH OOC OH O H CH 2 OH O H Cys130 His70 Tyr429 Asp355 Cys130 His70 Tyr429 – glucono-1,5-lactone H 2 O O 2 H 2 O 2 – -D-glucose FAD FAD FADH gluconic acid Tyr429 – – – 25 cellobiose dehydrogenase and BsCOX1 belong to the glutathione reductase (GR) superfamily. Location of the active center at the re face of the isoalloxazine ring is generally conserved within the GR superfamily, whereas that of the PCMH members is on the si side. Therefore, these structures provide elegant examples of convergent evolution, where starting from different ancestral folds, the same FAD-assisted glucose (or Fig. 2. (a) The GOOX structure. The protein consists of a FAD-binding (F) and a substrate-binding (S) domain, colored in red (helix) and green (strand). The intermediate analogue ABL ( cyan ), the cofactor FAD ( black ) and the linking residues His70 and Cys130, and the glycosylated Asn305 and Asn341 are displayed as ball-and- stick representations. The two major FAD-interacting segments are highlighted in magenta . (b) The 2Fo - Fc electron density map for the FAD cofactor and the covalently bound His70 and Cys130 contoured at 2 σ level. The density map demonstrates the first known double attachment flavinylation, 6- S - cysteinyl, 8 α - N 1-histidyl FAD. (b) (a) Fig. 3. (a) The interaction networks between ABL and GOOX. Hydrogen bonds are shown as green dashed lines. (b) Molecular surfaces of GOOX. The protein surface is colored for electrostatic potential from -20 k B T ( red ) to 20 k B T ( blue ), reflecting its pI value of 4.3-4.5. The FAD cofactor is colored in green , while the modeled cellobiose is in y ellow . The open carbohydrate-binding groove explains why oligosaccharides are good substrates. (a) (b) References [1] M.H. Lee et al. : Appl. Environ. Microbiol. 71 (2005) 8881. [2] C.-H. Huang, W.-L. Lai, M.-H. Lee, C.-J. Chen, A. Vasella, Y.-C. Tsai and S.-H. Liaw: J. Biol. Chem. 280 (2005) 38831. [3] M. Mewies et al. : Protein Sci. 7 (1998) 7. [4] O. Dym & D. Eisenberg: Protein Sci. 10 (2001) 1712. Shwu-Huey Liaw The Structural Biology Program, National Yang-Ming University, Taiwan E-mail: shliaw@ym.edu.tw cholesterol) oxidation is achieved through opposite flavin faces within distinct substrate-binding sites. Even starting from a similar structural fold, the sugar oxidases have evolved a similar FAD-assisted oxidation mechanism but different substrate recognition, resulting in distinct binding affinities to the mono- and di-sacchardes, with Km values ranging from 50 μ M to 30 mM.
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