|Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
Gene cluster involved in biosynthesis of pyrroloquinoline quinone, PQQ, had been determined as pqqABCDEFG. The mutant defected in pqqC produced a biosynthetic intermediate, and it was converted to PQQ in vitro by PqqC protein, which was expressed in Escherichia coli transformant and purified. This intermediate was purified and its structure was confirmed as 3a-(2-amino-2-carboxy-ethyl)-4,5-dioxio-4,5,6,7,8,9-hexahydro-quinone-7,9-dicarboxylic acid. The PqqC enzyme reaction required a protein factor (ActF) plus NADPH to show full enzyme activity, however, dithiothreitol (DTT) itself could be replaced. When mixed with PQQ, PqqC/D, a fusion protein of PqqC and PqqD, was eluted as a complex in gel filtration column chromatography, but separated each other in the presence of DTT. In the case of PqqC, part of the complex remained even in the presence of DTT. DTT has an ability to reduce PQQ, thus PqqC and PqqC/D might bind the oxidized PQQ tightly but not the reduced form so much. Association constant of PqqC or PqqC/D with PQQ was determined by measuring fluorescent quenching of the protein with titration of PQQ. Partially purified ActF showed NADPH oxidase activity in the presence of PQQ. It is suggested that DTT or ActF plus NADPH keeps PQQ in the reduced form and consequently enhances PQQ production by PqqC or PqqC/D in vitro. PqqD might work to lower the affinity of PqqC to the reduced form of PQQ. PqqE was expressed in E.coli and purified in the presence of DTT. When DTT was absent, PqqE seemed easy to be degraded. It is suggested that PqqE contained iron-sulfur cluster according to its absorption spectrum.