PURIFICATION AND ANALYSIS OF A FLAVOPROTEIN FUNCTIONAL AS NADH OXIDASE FROM AMPHIBACILLUS-XYLANUS OVEREXPRESSED IN ESCHERICHIA-COLI

The gene encoding the Amphibacillus xylanus flavoprotein has been clon ed into pTTQ18 and overexpressed in Escherichia coli, The recombinant enzyme has been purified to homogeneity yielding 15 mg of pure enzyme/ liter liter of cell culture. Recombinant flavoprotein is fully active and has an absorption spectrum identical to that of the enzyme purifie d from A. xylanus. The N-terminal sequence analysis and analytical gel filtration data confirm the structural identity of recombinant and A. xylanus enzymes. The K-m value for oxygen and the K-m value for NADH are 1.7 mM and 33.3 mu M, respectively. In the presence of free additi onal FAD, however, the K-m value for oxygen decrease dramatically. The NADH oxidase activity is accelerated markedly in the presence of addi tional FAD. The intracellular free FAD concentration of A. xylanus is calculated about 13 mu M. This FAD concentration would be enough to ac celerate the NADH oxidase activity of flavoprotein in cells of A. xyla nus. Two-electron reduction of the enzyme FAD by the strong reductant dithionite occurs during the total uptake of 6 electrons. Such behavio r usually indicates the presence of non-flavin redox centers. The high degree of homology between this enzyme and alkyl hydroperoxide reduct ase F52a protein and thioredoxin reductase suggests that these centers are the redox-active disulfide adjacent to the FAD and another disulf ide, which is able to slowly interchange with the redox-active disulfi de. The presence of two disulfides has been demonstrated.