Osao Adachi

Method of enzymatic determination of pyrroloquinoline quinone

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.

Determination of seminal fructose using d-fructose dehydrogenase

Seminal plasma, the liquid component of semen, is mainly secreted from the seminal vesicles and is characterized by relatively high concentrations of fructose and extremely low concentrations of glucose [l]. Fructolysis is a main energy source for sperm. Thus, assay of seminal fructose can provide information concerning the activity of the seminal vesicles and the condition of the ejaculatory ducts [2]. Various disorders of these organs may be responsible for cases of infertility [a]. Determination of seminal fructose has been done by paper chromatography 131, gas chromatography [4], calorimetry [S] and enzymic methods 1671. We describe an enzymic method for seminal fructose using bacterial membranebound D-fructose dehydrogenase (E.C. 1.1.99.11) [8.9].

The 9-carboxyl group of pyrroloquinoline quinone, a novel prosthetic group, is essential in the formation of holoenzyme of D-glucose dehydrogenase

Availability of different analogues of pyrroloquinoline quinone as the prosthetic group for apo-D-glucose dehydrogenase was examined. The 9-carboxyl group of pyrroloquinoline quinone was shown to be essential for the reconstitution of the enzyme activity. Although the carboxyl group may not be involved in catalytic function, it is quite probable to contribute the binding of the prosthetic group to apoenzyme.

Membrane-Bound Dehydrogenases of Acetic Acid Bacteria

One of the major key features of acetic acid bacteria is their strong oxidation ability of alcohols and sugars, resulting in quantitative production of oxidized compounds. Respiratory chains consisting of ubiquinone, terminal ubiquinol oxidase, and several primary membrane-bound dehydrogenases are responsible for this unique ability. Here, we describe recent progress in the understanding of enzymatic and molecular properties and biogenesis of the membrane-bound dehydrogenases, such as pyrroloquinoline quinone-dependent alcohol dehydrogenase–cytochrome complex, and recent findings on new membrane-bound dehydrogenases. Quinate oxidation by quinate dehydrogenase (QDH) of acetic acid bacteria is a key agent in the vitro shikimate production process composed of the membranes containing QDH and 3-dehydroquinate dehydratase and NADP+-dependent shikimate dehydrogenase. The addition of a catalytic amount of NADP+ and an NADPH-regeneration system drive the process forward to produce shikimate with almost 100 % yield. The pentose oxidation respiratory chain produces 4-keto-d-arabonate or 4-keto-d-ribonate, depending on the substrate. Novel three different membrane-bound enzymes are indicated: d-aldopentose 4-dehydrogenase, 4-keto-d-aldopentose 1-dehydrogenase, and d-pentonate 4-dehydrogenase.