Rare Keto-Aldoses from Enzymatic Oxidation: Substrates and Oxidation Products of Pyranose 2-Oxidase

Abstract

Pyranose oxidases are known to oxidise d-glucose, d-xylose and lsorbose to keto-aldoses, biochemically interesting compounds that may also be used for synthetic purposes in a variety of reactions. In this study pyranose oxidase from the basidiomycete Peniophora gigantea was investigated, and it was found that this enzyme is able to oxidise a broad variety of substrates very effectively. In analogy to its natural mode of action, most substrates are oxidised regioselectively in position 2. Certain compounds, however, are converted into 3-keto derivatives, and the enzyme even exhibits transfer potential, that is, disaccharides are formed from bglycosides of higher alcohols. Substrates that may be oxidised at C-2 in yields between 40 ± 98 % are d-allose, d-galactose, 6-deoxy-d-glucose, d-gentiobiose, a-d-glucopyranosyl fluoride and the very interesting 3-deoxy-d-glucose. 1,5-Anhydro-d-glucitol (1-deoxy-d-glucose) is very effectively oxidised in position 2 in 98 % yield and additionally gives a product of dioxidation at C-2 and C-3 upon prolonged reaction time. Selective oxidation at C-3 was found for 2-deoxyd-glucose in very good yields and for methyl b-d-gluco-and methyl b-galactopyranoside in lower yields. All oxidation products were unequivocally characterised by NMR spectroscopy and/or chemical derivatisation. In addition, the kinetic data of the enzymatic reactions were determined for all substrates. On the basis of these data and the structural characteristics of the substrates, a model for the minimal structural requirements of the enzyme ± substrate interaction is suggested. The enzyme presumably uses two different binding modes for the regioselective C-2 and the C-3 oxidations, which are described.

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

“…However, as discussed above, D-xylose is oxidized at C2 at an appreciable rate. For the P. gigantea enzyme, the O6 hydroxyl group has been dismissed as non-essential for regioselectivity (10); however, given the data presented in this work, it appears that the precise catalytic performance and substrate specificity differ between the two P2Ox enzymes.…”

“…Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7).…”

“…Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl ␤-D-glucosides (13,10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.…”

“…The reaction mechanism of P2Ox is of the type Ping Pong Bi Bi typically found in flavoprotein oxidoreductases (15,16). In the reductive half-reaction, an aldopyranose substrate reduces the FAD cofactor to yield reduced flavin (FADH 2 ) and 2-ketoaldose (aldos-2-ulose) as a result of oxidation at position C2 of the sugar substrate (1,10). The ensuing oxidative half-reaction involves the re-oxidation of FADH 2 …”

l-Glucitol Catabolism inStenotrophomonas maltophiliaAc

“…NMR spectra were recorded in D 2 O on a Bruker AMX 500 spectrometer at 500.14 MHz for 1 H and 125.76 MHz for 13 C at 300°K. Chemical shifts were referenced to internal acetone (␦ ϭ 2.030 and 30.50 ppm) (11,12).…”