Determination of PQQ-adduct with PQQ-liberating enzyme

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Two species of PQQ-serine were formed by incubating PQQ with serine. The ratio of these PQQ-serine was depend on pH of the reaction mixture. When PQQ-adducts formation with serine was conducted at pH 8.0, the simplest PQQ-oxazole, PQQ-glycine was given as sole PQQ-adduct. On the other hand, another species of PQQ-adduct was formed when incubated at pH 3.5. These two PQQ-adducts were stable themselves and were not interconvertible each other. As a result of structural analysis, the PQQ-serine prepared at pH 3.5 has a -CH_2OH group originated from serine. When the reaction of PQQ with various amino acids was carried out at pH 3.5, only one species of PQQ-adduct was formed, which are different in chemical structure from PQQ-glycine. Both species of PQQ-serine exhibited a marked growth stimulating activity for microorganisms.Growth stimulating effect of PQQ for E. coli was clearly observed under well defined conditions. This PQQ effect was classfied to the type II effect. When E. coli grew on a medium containing PQQ, about 50% of whole GDH was converted to holoenzyme during cultivation. At this moment, E. coli consumed a large amount of glucose and the medium was highly acidified more than in a medium excluding PQQ. From these results, GDH oxidizes glucose to gluconate but gluconate was scarecely taken up into the cell. GDH functions to generate proton gradient, followed by ATP synthesis. The ATP supports glucose uptake via PTS, subsequently the growth of E. coli is stimulated. Apo-GDH is widely distributed in microorganisms, thus, this PQQ effect is estimated to be an universal phenomenon. A proposed mechanism of growth stimulating effect of PQQ and PQQ-adducts for microorganism is discussed.