Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

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The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the ␣/␤ hydrolase-fold topology. The catalytic residues, Asp 107 and His 275 , are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His 275 . Because of a crystallographic contact, the active site has become accessible for the Gln 134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr 152 /Tyr 215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.Epoxide hydrolases (EC 3.3.2.3) are a group of functionally related enzymes that catalyze the cofactor-independent hydrolysis of epoxides to their corresponding diols by the addition of a water molecule. Epoxides are very reactive electrophilic compounds frequently found as intermediates in the catabolic pathway of various xenobiotics. For instance they are the carcinogens formed by bioactivation reactions catalyzed by cytochrome P450. Therefore, conversion of epoxides to less toxic, watersoluble compounds is an essential detoxification step in living cells. Consequently, epoxide hydrolases have been found in a wide variety of organisms, including mammals, invertebrates, plants, and bacteria (1).Until now most research has been focused on mammalian epoxide hydrolases (2, 3), which, together with glutathione S-transferases, are the most important enzymes to convert toxic epoxides to more polar and easily excretable compounds (4). However, much progress has recently also been made in the characterization of bacterial epoxide hydrolases (5, 6, 7). These enzymes show a significant sequence homology with those of mammalian origin. They can be easily obtained in large amounts, and they exhibit enantioselectivity with various industrially important epoxides, which makes them promising biocatalysts for the large scale preparation of enantiopure epoxides and/or their corresponding vicinal diols (8). In particular, extensive studies have been performed on the epoxide hydrolase from Agrobacterium radiobacter AD1, a Gram-negative bacterium that is able to use the environmental pollutant epichlorohydrin as its sole carbon and energy source (5,6,8). This epoxide hydrolase is a soluble monomeric globular protein of 35 kDa with a broad substrate range. Epichlorohydrin and epibromohydrin are its best substrates, and the optimum pH range for catalysis...