Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes.

Abstract

Although they have been studied for nearly 50 years, the source of the FMNH2 needed for effective biooxidation by the 2,5- and 3,6-diketocamphane monooxygenase (DKCMO) isoenzymes induced by the growth of Pseudomonas putida NCIMB 10007 (ATCC 17453) on camphor remains incompletely characterized. Prior studies have focussed exclusively on enzymes present in cells harvested during late-exponential-phase growth despite considerable circumstantial evidence that the flavin reductase (FR) component of these multicomponent monooxygenases is subject to growth-phase-dependent variation. In this study, a number of alternative FMNH2-generating activities, including both conventional FRs and enzymes also able to serve as ferric reductases, were isolated from camphor-grown cells, and the relative level, and hence potential contribution, of these various proteins shown to vary considerably depending on the point of harvest of NCIMB 10007 within exponential-phase growth. While two constitutive monomeric ferric reductases (molecular masses 27.0 and 28.5 kDa) were found to be the major relevant sources of FMNH2 during the initial stages of growth on camphor-based media, a significant subsequent contribution throughout the mid- to late-exponential phases of growth was also made by the camphor-induced homodimeric 37.0 kDa FR Fred, recently reported to serve such a role exclusively. The possible involvement of camphor-induced putidaredoxin reductase (51.0 kDa) as a contributory activity was also investigated and considered. Studies with highly purified preparations of the isofunctional DKCMOs confirmed the potential of the various reductases to function effectively as sources of the requisite FMNH2 to both monooxygenases at different times throughout growth on camphor.