Membrane-Bound Pyrroloquinoline Quinone-Dependent Dehydrogenase in Gluconobacter oxydans M5, Responsible for Production of 6-(2-Hydroxyethyl) Amino-6-Deoxy- l -Sorbose

Abstract

A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent D-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.Gluconobacter strains are able to oxidize many sugar alcohols incompletely to produce the corresponding aldehydes, ketones, and organic acids, e.g., L-ascorbic acid, D-gluconic acid, ketogluconic acids, and dihydroxyacetone. Their responsible polyol dehydrogenases have been confirmed (1,4,15). It was reported that Gluconobacter oxydans strain M5 can catalyze the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), a precursor of the antidiabetic drug miglitol (11,20). However, which dehydrogenase is involved has not been revealed yet. The aim of this work was to find out the responsible enzyme.The strains and plasmids used in this study are listed in Table 1. G. oxydans M5 was grown in a medium containing 20 g of D-sorbitol, 3 g of yeast extract, 10 g of polypeptone, 1 g of KH 2 PO 4 , and 0.2 g of MgSO 4 -7H 2 O in 1 liter of deionized water. The different fractions of the cells were prepared by supercentrifugation at 100,000 ϫ g for 60 min. The activities of 1NSL oxidation were measured in the presence of 200 mM substrate, 1NSL (8). The substrate and product were analyzed by silica gel thin-layer chromatography (TLC), with ethanolmethanol-ammonia (1.5:1:1 [vol/vol/vol]) as its eluant, and then dyed with iodine. The major part of the activities was found in the membrane fraction, but little activity could be found in the cytoplasmic fraction (Fig. 1). A second experiment showed phenazine methosulfate (PMS)-dependent 1NSL dehydrogenase activity but not NAD(P)-dependent 1NSL dehydrogenase activity in the membrane fraction. On the contrary, the cytoplasmic fraction had only NAD(P)-dependent 1NSL dehydrogenase activity. In cell extracts, the total activity of PMS-dependent 1NSL dehydrogenase was some 28-fold higher than that of the NAD(P)-dependent 1NSL dehydrogenase (data not shown). These results suggested that the proteins responsible for 1NSL oxidation in G. oxydans are located mainly in the cytoplasmic membrane.Gluconobacter strains contain a large number of dehydrogenases, which can be classified into two major groups (15). The first group is formed by cytoplasmic-soluble NAD(P)-dependent polyol dehydrogenases, which were believed to participate in the synthesis of precursors and are obviously involved in the maintenance of cells in the stationary growth phase (12, 15). The second group possesses various membranebound dehydrogenases, which are shown to be responsible for the rapid oxidation of some important substrates (16,17). According to our previous work, the oxidation of 1NSL to 6NSE by G. oxydans M5 was fast, with almost 95%...

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster

“…Bacterial strains, plasmids and cultivation G. oxydans M5 was selected in our laboratory to produce L-sorbose from D-sorbitol [15]. The pMD19-T kit was purchased from TaKaRa.…”

Characterization of Enzymes in the Oxidation of 1,2-Propanediol to d-(−)-Lactic Acid by Gluconobacter oxydans DSM 2003

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Revealing in vivo glucose utilization of Gluconobacter oxydans 621H Δmgdh strain by mutagenesis

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