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Glucose oxidase is a flavin-dependent enzyme which catalyses the oxidation of beta-D-glucose by molecular oxygen to delta-gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously refined at 2.3 A resolution, has been refined at 1.9 A resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and refined at 1.8 A resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 A for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force-field methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the beta-D-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to flavin adenine dinucleotide. Other hexoses, such as alpha-D-glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-D-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, delta-gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.

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The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. Keywords : glucose oxidase; Penicillium amagasakiense ; amino acid sequence; glucose-methanol-choline oxidoreductase ; consensus pattern.The glycoprotein glucose oxidase catalyses the oxidation of stable but has a sixfold higher affinity for β-D-glucose and a β-D-glucose to D-glucono-1,5-lactone with the concomitant re-tenfold higher specificity constant than glucose oxidase from duction of molecular oxygen to hydrogen peroxide. The enzyme Aspergillus niger (Kalisz et al., 1997). Despite the commercial has been purified from Aspergillus (Pazur and Kleppe, 1964; importance and extensive industrial application of glucose oxiSwoboda and Massey, 1965) and Penicillium species (Kusai et dase (Röhr et al., 1983;Crueger and Crueger, 1984; Wilson and al., 1960). Fungal glucose oxidase is a homodimer of approxi-Turner, 1992), and the availability of the preliminary X-ray difmately 160 kDa containing two tightly, but non-covalently, fraction data , the three-dimensional strucbound FAD cofactors (Yoshimura and Isemura, 1971a,b; Hay-ture of P. amagasakiense glucose oxidase has only recently been ashi and Nakamura, 1976) and 13% carbohydrate of the high elucidated (Wohlfahrt, G., Witt, S., Herdle, J., Kalisz, H. M., mannose type (Hayashi and Nakamura, 1976; Kalisz et al., Schomburg, D. and Hecht, H. J., unpublished results). The main 1997). Glucose oxidase from Penicillium amagasakiense is less stumbling block was the unavailability of its primary structure.In contrast, both the amino acid sequence (Kriechbaum et al., Correspondence to H. M. Kalisz, Gesellschaft für Biotechnologische 1989; Frederick et al., 1990) and X-ray structure (Hecht et al.,

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Tryparedoxin, a thioredoxin-related protein from Crirhidiu jusciculutu with a molecular mass of 16 kDa catalyses the reduction of a peroxiredoxin-type peroxidase, Cf21, at the expense of trypanothione [Nogoceke, E., Gommel, D. U., KieR, M., Kalisz, H. M. & FlohC, L. E. (1997) Biol. , The kinetic analysis of tryparedoxin revealed an enzyme substitution mechanism. The corresponding molecular event was elucidated to be a reversible oxidoreduction of the disulfide bridge in the thioredoxin-related motif WCPPC. The amino-proximal cysteine residue of this active site was more reactive in S-alkylation experiments than the distal residue. The natural substrates of tryparedoxin, trypanothione and Cf21, could only be substituted by glutathione and glutathione disulfide with considerable loss in activity. The pronounced specificity of tryparedoxin is further accentuated by low limiting K,, values for Cf21 and trypanothione (2.2 pM and 130 pM, respectively, as compared to 990 pM for gluthathione disulfide and an infinite value for glutathione). Tryparedoxin can therefore be classified as a trypanothione: peroxiredoxin oxidoreductase. The reduction of tryparedoxin by trypanothione appears to be the rate-limiting step in the trypanothione-dependent hydroperoxide reduction because(a) the regeneration of reduced tryparedoxin from the tryparedoxin-trypanothione complex is rate limiting (k,,l, 392 min-'), (b) the physiological trypanothione concentrations may not always saturate tryparedoxin, and (c) the rate constants for the net forward reaction of Cf21 are faster than those of the tryparedoxin reaction. The functional characteristics of tryparedoxin explain the limited capacity of trypanosomatids in coping with oxidative stress and qualify the enzyme as a potential target for the design of specific trypanocidal compounds.