Soluble glucose dehydrogenase (s-GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ-dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X-ray structure of s-GDH with the cofactor at 2.2 A resolution, and of a complex with reduced PQQ and glucose at 1.9 A resolution. These structures reveal the active site of s-GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ-dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base-catalyzed hydride transfer, rather than the generally accepted covalent addition-elimination mechanism. Thus, PQQ-dependent enzymes use a mechanism similar to that of nicotinamide- and flavin-dependent oxidoreductases.

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The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH-and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.In nature, many oxygenation reactions are carried out by flavin-dependent monooxygenases (24). The diversity of conversions that can be catalyzed is large; the reactions include Baeyer-Villiger reactions, aromatic hydroxylations, sulfoxidations, amine oxidations, and epoxidations. Many of these conversions occur with high enantio-and/or regioselectivity. The variety of reactivity is also reflected in the diverse physiological processes in which these enzymes play a prominent role, including xenobiotic compound metabolism in humans (49), biosynthesis of toxins (21), and pollutant degradation by bacteria (46).Enantio-and regioselective oxygenations are often difficult to achieve by chemical means, while these types of reactions can lead to valuable optically active compounds. Due to their exquisite regio-and/or enantioselectivity and catalytic efficiency, flavin-dependent monooxygenases are useful biocatalysts for the synthesis of a variety of fine chemicals (12,35,41,47). However, so far, only a limited number of flavin-dependent monooxygenase genes have been cloned and overexpressed, which has limited the application of these biocatalysts for synthetic processes.The Baeyer-Villiger reaction, i.e., the oxidation of ketones or aldehydes by peroxides resulting in oxygen insertion adjacent to the carbonyl group (2), has many applications in organic ...

Kinetic and optical studies were performed on the reductive half-reaction of soluble, quinoprotein glucose dehydrogenase (sGDH), i.e., on the conversion of sGDHox plus aldose sugar into sGDHred plus corresponding aldonolactone. It appears that the nature and stereochemical configuration of the substituents at certain positions in the aldose molecule determine the substrate specificity pattern: absolute specificity exists with respect to the C1-position (only sugars being oxidized which have the same configuration of the H/OH substituents at this site as the beta-anomer of glucose, not those with the opposite one) and with respect to the overall conformation of the sugar molecule (sugars with a 4C1 chair conformation are substrates, those with a 1C4 one are not); the nature and configuration of the substituents at the 3-position are hardly relevant for activity, and an equatorial pyranose group at the 4-position exhibits only aspecific hindering of the binding of the aldose moiety of a disaccharide. The pH optimum determined for glucose oxidation appeared to be 7.0, implying that reoxidation of sGDHred is rate-limiting with those electron acceptors displaying a different value under steady-state conditions. The kinetic mechanism of sGDH consists of (a) step(s) in which a fluorescing intermediate is formed, and a subsequent, irreversible step, determining the overall rate of the reductive half-reaction. The consequences of this for the likeliness of chemical mechanisms where glucose is oxidized by covalent catalysis in which a C5-adduct of glucose and PQQ are involved, or by hydride transfer from glucose to PQQ, followed by tautomerization of C5-reduced PQQ to PQQH2, are discussed. The negative cooperative behavior of sGDH seems to be due to substrate-occupation-dependent subunit interaction in the dimeric enzyme molecule, leading to a large increase of the turnover rate under saturating conditions.

Glucose electrodes were prepared by "wiring" quinoprotein glucose dehydrogenase, GDH (EC 1.1.99.17) to glassy carbon with an osmium complex containing redox-conducting epoxy network. Their current density at 70 mM glucose concentration reached 1.8 mA cm-2 when 15 #tg cm-2 of the enzyme having an activity of 250 units mg-1 was applied to the electrode. Under the same conditions, electrodes made with glucose oxidase (GOX) of similar activity (250 units mg-1) had a maximum current density of 0.88 mA cm-2. The maximum current density was reached with 8 % GDH In the redox polymer film. The current density was almost flat through the 6.3-8.8 pH range and was not altered when the solution was either aerated or argon purged. It decreased at 25 °C to half Its Initial value In 8 h.