Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD؉ , divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both ␣-and ␤-specific enzymes. The ␣-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD ؉ and Mn 2؉ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD ؉ and maltose, refined at a resolution of 1.9 Å. The NAD ؉ is bound to a typical Rossman fold NAD ؉ -binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme.

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Replacement of the cyclohexane chair-form rings in the vertical planes of the diamond lattice by boat-form rings results in the wurzite lattice. If this is also carried out on the horizontal planes, one obtains an unknown carbon modification,l]' whose smallest characteristic structural unit, the tetrasterane 1, is known.lzl Earlier, we succeeded in enlarging 1 by the addition of two cyclohexane rings to give 2.13] We now report the synthesis of the double tetraasterane 9141 (Scheme 1). 2Photoexcitation of 1,4,5,8-tetrahydronaphthalene-2,3-dicarboxylic anhydride 3[5i in dioxane with a high-pressure mercury lamp equipped with a Solidex filter in the presence of benzophenone as photosensitizer results in the formation, in approximately 8% yield, of a sparingly soluble mixture consisting of at least four dimers. Fractional sublimation of this mixture at torr gave a forerun at about 250°C followed by the desired dimer 4 starting at 310°C (relative amount in the mixture, 46%; m.p. >350°C). Compound 4 was treated with methanol/acetyl chloride to convert it into the tetramethyl ester 6 ( Table I). The anti head-head dimer 5 undergoes sublimation already at 190-200°C and was likewise characterized as the tetramethyl ester (10). The structures of 6 and 10 were confirmed by X-ray crystal structure analysi~.[~.~I Chromatography of the mother liquors of 10 on SiOz revealed the presence of two additional compounds. The faster eluting component was [**I Asteranes, Part 19. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We thank A. Kuiper. P. Lung. 1. M a i m I . Rossnagel, I . Suss. and U. Tanger for numerous spectra, gas chromatograms, and elemental analyses.-Part 18: A. Otterbach, H. Musso, Angew Chem. 99 (1987) 588; Angew. Chem. Inr. Ed. Engl. 26 (1987) 554.isolated and identified spectroscopically as the doubly aromatized derivative of 10, namely, 12. The more slowly eluting component could not be obtained in pure form. The tetraasterapropellane structure of 11 can be derived from its 'H-NMR spectrum.RJcj EJt : R r 6 , R = C0,CH3 r8, 6 4 ' . R = = Co2H CI ' L9, R = H 10, R = C0,CH3 11 12 R = CO, CH, Scheme 1. a) 3 (6.8 g, 33.6 mmol), benzophenone (200 mg), dioxane (200 mL) under N2. Philips HPK (I25 W), Solidex filter, irradiated for 4 d. 560 mg (8%) 4 + 5 (see text). b) 4 (65 mg, 0.16 mmol), methanol (IOmL), acetyl chloride (1.0 mL), refluxed for 5 d, heterogeneous reaction, 69 mg (87%) 6, m.p.=324"C. c) 5 (200 mg, 0.48 mmol) was added to a solution containing sodium (50 mg) in methanol (15 mL) and the solution was acidified with dilute H,S04. The monoester was extracted with ether and then treated with diazomethane in ether, affording 124 mg (51%) 10, m.p.=230"C. d) 6 (400 mg, 0.99 mmol) dissolved in 2 N NaOH (50 mL) over 30 min. The solution was then acidified with 2 N H$04, resulting in the formation of a fine, colorless precipitate, 396 mg (90%) 7 . e) A suspension of 7 (690mg, 1.56 mrnol), N-chlorosuccinimide (3.22 g, 24 mmol), and Pb(OAc), (5.0 g, 6.3 mmol) in d...

VI v/v Volumen pro Volumen Vol. Volumen w/v Gewicht pro Volumen X-Gal Lactose, Sucrose, Galaktose, Maltose und Xylose werden verwertet, wobei nach Wachstum auf Glukose die Fermentationsprodukte H 2 , CO 2 , Acetat und Ethanol gefunden wurden. Xylan und Cellulose können nicht verwertet werden (Davey et al., 1993). P. miotherma toleriert im Gegensatz zu T. maritima zwischen 2% und 10% NaCl. P. miotherma besitzt die Fähigkeit, elementaren Schwefel zu H 2 S zu reduzieren 1.1.1. Adaptation an extreme Habitate Abbildung 1: Auf 16S rRNA beruhender phylogenetischer Stammbaum. Die Positionen hyperthermophiler Organismen sind mit dicken Linien gekennzeichnet. (nach Selig et al., 1997, modifiziert)

The title compound (IIIc) is prepared starting from the anhydride (I).