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 FMNH 2 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 FMNH 2 -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 FMNH 2 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 FMNH 2 to both monooxygenases at different times throughout growth on camphor.

Flavin-Dependent Redox Transfers by the Two-Component Diketocamphane Monooxygenases of Camphor-Grown Pseudomonas putida NCIMB 10007

“…The other inducible FNR-generating activity was located exclusively in relevant low MW fractions, and was only detectible in significant amounts from around the mid-log phase of camphor-dependent growth. Using previously described protocols [15], purification and subsequent characterisation confirmed that the principal protein in relevant low MW fractions corresponded to Fred, the chromosomally-coded 36 kDa NADH-dependent FNR-generating homodimeric enzyme isolated previously from camphor-grown P. putida NCIMB 10007 [5]. …”

“…One of these activities, located exclusively in the high MW fractions as confirmed by relevant native PAGE gels, promoted putidaredoxin-linked reduction of electron acceptors at the expense of NADH, and was induced in parallel with camphor exo -hydroxylase (Supplementary Figure S1). The principal protein present in the relevant samples was isolated (~51.0 kDa), analysed, and thereby confirmed to be identical to PdR [15], the monomeric protein product of the camA gene with a calculated molecular mass of 48.5 kDa [37,38]. The other inducible FNR-generating activity was located exclusively in relevant low MW fractions, and was only detectible in significant amounts from around the mid-log phase of camphor-dependent growth.…”

“…Samples of highly purified Fred were prepared at 4 °C using an LKB Biologic FPLC system deploying the three-stage protocol fully described by Willetts and Kelly [15]. A combined Frp1 plus Frp2 preparation was a by-product of the procedure, and this was resolved into separate highly purified Frp1 and Frp2 activities using the purification protocol developed and fully described by Halle and Meyer [30].…”

“…Enzyme activities were routinely assayed spectrophotometrically (Hewlett-Packard model 8452A, Hewlett-Packard, Palo Alto, CA, USA) under anaerobic conditions by measuring the initial rate of the decrease in absorbance at 340 nm as described previously [15]. For Frp1, Frp2 and Fred, each respective 3.0 mL reaction mixture containing 60 mM Tris/HCl buffer (pH 7.6), 10–100 μM NADH, and 2–30 μM FMN was placed in a cuvette (3.5 mL, 1 cm light path) equipped with a magnetic stirrer bar and rubber septum.…”

“…Taking the ability to promote NADH-dependent biooxidation of ketones to lactones by highly purified preparations of DKCMOs as the relevant parameter, it was subsequently further confirmed [15] that native FNR-generating enzymes other than Fred could serve such a role for the DKCMOs with equal or even greater efficiency during growth of P. putida NCIMB 10007 on camphor-based defined media. The principal relevant activities were shown to be two constitutive ferric reductases, chromosome-coded enzymes which have been shown previously to serve as FRs able to generate freely diffusible FNR in P. putida KT2440 [16,17].…”