Putidaredoxin Reductase Research Articles

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Transition of stability of putidaredoxin reductase by introducing proline

The monooxygenase activity of the Pseudomonas putida cytochrome P450 system was expressed in the presence of P450cam, putidaredoxin reductase (PdR), and putidaredoxin (Pd). In turn, it has been found to catalyze the oxidation of various organic compounds. Since cytochrome P450 system substrates are often insoluble in water, reactions should be carried out at high temperatures and in the presence of organic solvents where the high stability of all of P450cam, PdR, and Pd are needed. In this study, proline was introduced to improve the stability of PdR which was estimated to have the lowest organic solvent stability compared to others. Thus, the half-life of PdR_T221P at 40°C was 1.37 times longer than that of wild-type PdR. However, the half-life of PdR_T221P in the presence of 25% (v/v) methanol was shorter than that of wild-type PdR. Furthermore, structural stabilities of PdRs using molecular dynamics (MD) simulations demonstrated these phenomena.

Bioorganometallic Chemistry at the Interface with Biocatalysis: Chemoselective Reduction of Biomimetic NAD+ Cofactors with [Cp*Rh(bpy)H]+, Tandem Catalysis with 1,4-NADH-Dependent Enzymes, Chiral Synthesis, Organotin Metabolites, and DFT Mechanism Studies

This Review will focus on our studies of the utilization of the [Cp*Rh(bpy)H]+ complex as a biomimetic hydrogenase, regeneration reagent for chemoselective reduction of biomimetic NAD+ cofactors, N-benzylnicotinamide triflate, 1, β-nicotinamide ribose-5′-methyl phosphate, 2, and natural NAD+, 3, in the conversion to their 1,4-NADH analogues, 1,4-dihydro-N-benzylnicotinamide, 4, and β-1,4-dihydronicotinamide-5′-ribose methyl phosphate, 5, and natural 1,4-NADH, 6, respectively. This regeneration reaction with 1,4-NADH biomimetics 4 and 5 was used in tandem with 1,4-NADH-dependent enzymes for organic synthesis. The enzyme examples will encompass horse liver alcohol dehydrogenase (HLADH) reductions of prochiral ketones to chiral S-alcohols; cytochrome P450 enzymes for selective C–H oxidation reactions of organic compounds, and organometallic substrates, tributyltin acetate and cyclohexyltriphenyltin, with 1,4-NAD(P)H; and the biomimetic 1,4-NADH cofactor, 4, with the 2-hydroxybiphenyl-3-monooxygenase enzyme, HbpA, a FAD-containing monooxygenase enzyme, for hydroxylation of ortho-substituted phenols to catechols. This Review also includes discussions on in-depth DFT mechanism studies, and other chemical mechanistic aspects, of all tandem catalytic reactions that demonstrate, utilizing a simplified model of the HLADH-Zn(II)-mediated enzyme, the unprecedented role of Zn(II) in the hydride transfer reaction. Moreover, site-directed mutagenesis with 1,4-NAD(P)H-dependent cytochrome P450 BM-3 improved the molecular recognition process for biomimetic 1,4-NADH cofactors in organic compound C–H oxidation reactions. The 1,4-NADH-dependent cytochrome P450 CAM recognized the biomimetic 1,4-NADH cofactors; however, site-directed mutagenesis of the putidaredoxin reductase (PdR) component increased the kinetics for the C–H oxidation reaction of camphor. Cyclohexyltriphenyltin demonstrated the differences in hydroxylation regioselectivity, when comparing the cytochrome P450 enzyme with biomimetic P450 porphyrin Fe(III) and Mn(III) models. The biomimetic 1,4-NADH cofactor, 1,4-dihydro-N-benzylnicotinamide, 4, was also utilized as a reducing agent, providing the reduced, chemoselective coenzyme, the FADH– anion, that further reacts with O2/H+ to provide the 4α-hydroperoxide flavin, for the HbpA enzymatic oxidative conversion of ortho-substituted phenols to catechols.

Engineering of Microbial Substrate Promiscuous CYP105A5 for Improving the Flavonoid Hydroxylation

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant’s kcat/Km values and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols.

CYP108N12 initiates p-cymene biodegradation in Rhodococcus globerulus

Rhodococcus globerulus (R. globerulus) isolated from soil beneath Eucalyptus sp. was found to live on the monoterpenes 1,8-cineole, p-cymene and (R)- and (S)-limonene as sole sources of carbon and energy. Previous metabolic studies revealed that R. globerulus is capable of living on 1,8-cineole, the main monoterpene component of eucalyptus essential oil through the activity of cytochrome P450cin (CYP176A1) [1]. Genomic sequencing of R. globerulus revealed a novel putative cytochrome P450 (CYP108N12) that shares 48% sequence identity with CYP108A1 (P450terp) from Pseudomonas sp., an α-terpineol hydroxylase. Given the sequence similarity between CYP108N12 and P450terp, it was hypothesised that CYP108N12 may be responsible for initiating the biodegradation of a monoterpene structurally similar to α-terpineol such as (R)-limonene, (S)-limonene or p-cymene. Encoded within the operon containing CYP108N12 were two putative bacterial P450 redox partners and putative alcohol and aldehyde dehydrogenases, suggesting a complete catalytic system for activating these monoterpenes. Binding studies revealed that p-cymene and (R)- and (S)-limonene all bound tightly to CYP108N12 but α-terpineol did not. A catalytically active system was reconstituted using the non-native redox partner putidaredoxin and putidaredoxin reductase that act with CYP101A1 (P450cam) from Pseudomonas. This reconstituted system catalysed the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol, and (R)- and (S)-limonene to (R)- and (S)-perillyl alcohol, respectively. R. globerulus was successfully grown on solely p-cymene, (R)-limonene or (S)-limonene. CYP108N12 was detected when R. globerulus was grown on p-cymene, but not either limonene enantiomer. The native function of CYP108N12 is therefore proposed to be initiation of p-cymene biodegradation by methyl oxidation and is a potentially attractive biocatalyst capable of specific benzylic and allylic hydroxylation.

The 16α-Hydroxylation of Progesterone by Cytochrome P450 107X1 from Streptomyces avermitilis.

Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous heme-dependent enzymes that catalyze the monooxygenation of non-activated C-H bonds to modify the structure of the substrate. In this study, we heterologously expressed CYP107X1 from Streptomyces avermitilis and conducted in vitro substrate screening using the alternative redox partners putidaredoxin and putidaredoxin reductase. CYP107X1 catalyzed the 16α-hydroxylation of progesterone with regio- and stereoselectivity. The spectroscopic analyses showed that CYP107X1 bound progesterone with a relatively high Kd value of 65.3±38.9 μM. The Km and kcat values for progesterone were estimated to be 47.7±12.0 μM and 0.30 min-1 , respectively. Furthermore, a crystal structure was obtained of CYP107X1 bound with glycerol from the buffer solution. Interestingly, a conserved threonine was replaced with asparagine in CYP107X1, indicating that it may adopt an unnatural proton transfer process and play a crucial role in its catalytic activity.

Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952.

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated KD value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min-1 nmol-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

Regio- and stereoselective hydroxylation of testosterone by cytochrome P450 from Streptomyces griseus ATCC 13273

16α-Hydroxylated steroids are important precursors in the synthesis of high value steroidal drugs such as glucocorticoids in the pharmaceutical industry. In this study, an innovative biotechnological method based on enzymatic catalysis was presented for the high-efficient production of 16α-hydroxyl-testosterone. CYP154C, a cytochrome P450 from Streptomyces griseus ATCC 13273, was identified to carry out a one-step regio- and stereoselective hydroxylation of testosterone along with putidaredoxin reductase (PDR) and putidaredoxin (Pdx) as the redox partner. The K m and k cat values of CYP154C for testosterone were 23.40 ± 3.69 μM and 4.75 min−1, respectively. The catalytic efficiency (k cat/K m) for testosterone was 12.18 s−1·μM−1. Using a CYP154-based whole-cell system in E. coli BL21(DE3), high efficiency of hydroxylation of testosterone for production of 16α-hydroxyl-testosterone was achieved. The bioconversion rate of testosterone (50 mg/L) by the whole cell system came up to 75 ± 2.87% within 24 h. These results demonstrated the high potential of CYP154-based biocatalytic system for 16α-hydroxyl-testosterone production.

Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis

Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C–H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners—RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The Km and kcat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min−1, and kcat/Km was 0.81 min−1 mM−1. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.

Conferring the Metabolic Self-Sufficiency of the CAM Plasmid of Pseudomonas putida ATCC 17453: The Key Role of Putidaredoxin Reductase.

The relative importance of camphor (CAM) plasmid-coded putidaredoxin reductase (PdR) and the chromosome-coded flavin reductases Frp1, Frp2 and Fred for supplying reduced FMN (FNR) to the enantiocomplementary 2,5- and 3,6-diketocamphane monooxygenases (DKCMOs) that are essential for the growth of Pseudomonas putida ATCC 17453 on (rac)-camphor was examined. By undertaking studies in the time window prior to the induction of Fred, and selectively inhibiting Frp1 and 2 with Zn2+, it was confirmed that PdR could serve as the sole active supplier of FNR to the DKCMOs. This establishes for the first time that the CAM plasmid can function as an autonomous extrachromosomal genetic element able to express all the enzymes and redox factors necessary to ensure entry of the C10 bicyclic terpene into the central pathways of metabolism via isobutyryl-CoA.

Specific N-demethylation of verapamil by cytochrome P450 from Streptomyces griseus ATCC 13273.

Norverapamil, the N-demethylated derivative of verapamil, is a novel promising leading compound for attenuating multidrug resistance with less side effects compared with verapamil. However, the efficient synthetic method for norverapamil is absent. In this study, an innovative biotechnological method based on enzymatic catalysis was presented for the high-efficient production of norverapamil. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, was identified to carry out a one-step specific N-demethylation of verapamil along with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) as the redox partner. Docking calculations rationalized the specific N-demethylation observed in experiment and identified important amino acid residues for verapamil binding. Furthermore, a CYP105D1-based whole-cell system in E. coli BL21(DE3) was established and optimized for highly efficient N-demethylation of verapamil. The bioconversion rate of verapamil by the whole cell system came up to 60.16% within 24 hours under the optimized conditions. These results demonstrated the high potential of CYP105D1-based biocatalytic system for norverapamil production.

Enzymatic Synthesis of ω-Hydroxydodecanoic Acid By Employing a Cytochrome P450 from Limnobacter sp. 105 MED

ω-Hydroxylated fatty acids are valuable and versatile building blocks for the production of various adhesives, lubricants, cosmetic intermediates, etc. The biosynthesis of ω-hydroxydodecanoic acid from vegetable oils is one of the important green pathways for their chemical-based synthesis. In the present study, the novel monooxygenase CYP153AL.m from Limnobacter sp. 105 MED was used for the whole-cell biotransformations. We constructed three-component system that was comprised of CYP153AL.m, putidaredoxin and putidaredoxin reductase from Pseudomonas putida. This in vivo study demonstrated that CYP153AL.m is a powerful catalyst for the biosynthesis of ω-hydroxydodecanoic acid. Under optimized conditions, the application of a solid-state powdered substrate rather than a substrate dissolved in DMSO significantly enhanced the overall reaction titer of the process. By employing this efficient system, 2 g/L of 12-hydroxydodecanoic acid (12-OHDDA) was produced from 4 g/L of its corresponding fatty acid, which was namely dodecanoic acid. Furthermore, the system was extended to produce 3.28 g/L of 12-OHDDA using 4 g/L of substrate by introducing native redox partners. These results demonstrate the utility of CYP153AL.m-catalyzed biotransformations in the industrial production of 12-OHDDA and other valuable building blocks.

Bacterial CYP154C8 catalyzes carbon-carbon bond cleavage in steroids.

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C-C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR-based structural elucidation of reaction products reveals 21-hydroxyprednisone as the major product for prednisone, while the other product is identified as 1-dehydroadrenosterone obtained due to C-C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C-C bond cleavage products.

Enzyme-catalyzed regio-selective demethylation of papaverine by CYP105D1.

To investigate the regio-selective demethylation of papaverine by CYP105D1 and develop a whole-cell biocatalytic system for the preparative synthesis of 6-O-demethyl-papaverine. CYP105D1 from Streptomyces griseus ATCC 13273 was used for the regioselective demethylation of papaverine at C-6 using putidaredoxin reductase (PDR) and putidaredoxin (Pdx) as the electron transport system. The Km value of CYP105D1 towards papaverine was estimated to be 92.24μM. Furthermore, a CYP105D1-based whole-cell system was established in E. coli BL21(DE3). The whole cell biotransformation condition was optimized as 25°C, pH 7.5, 8g (cell dry weight) L-1 whole cell biomass and 3% (v/v) PEG-200 as cosolvent. Under the optimal condition, the conversion yield of papaverine reached to 61.15% within 24h. The selective demethylation of papaverine by CYP105D1 was accomplished. The CYP105D1-based whole-cell biocatalyst has a potential used for the efficient synthesis of 6-O-demethyl-papaverine.

A Photoenzymatic NADH Regeneration System.

A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD+ into the enzyme‐active 1,4‐NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution.

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO3+ ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H2 O2 , with optimum conversion surprisingly being achieved at ≈75 mm H2 O2 . More importantly, H2 O2 tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H2 O2 . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

Artificial Self-Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity.

Most bacterial cytochrome P450 monooxygenases (P450s) do not work alone because their active species is generated by two electrons supplied through two separate auxiliary proteins. Artificial "self-sufficient" P450s, in which one molecule each of the two auxiliary proteins is arranged close to the P450s, have been developed but have not achieved the maximum catalytic turnover numbers of the P450s. In this study, the Pseudomonas putida P450 (P450cam) is assembled with multiple molecules of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), by fusion to a heterotrimeric protein. In the assembled P450cam containing one PdX and one PdR, kinetic analysis reveales that the catalytic cycle of P450cam is suspended twice awaiting the reduction of PdX by PdR. An increase in the number of PdR molecules stimulated the PdX reduction process. Assembly with two PdXs allows one PdX to be reduced during the binding of the other PdX to P450cam for the first electron transfer, eliminating one waiting step. Finally, P450cam assembled with two PdXs and three PdRs showes 92% of the maximum activity of free P450cam. Therefore, assembly with multiple molecules of auxiliary proteins will facilitate in vitro biotechnological applications of the P450s.

A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of α-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of PnTPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450terp).

Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a direct microbial biosynthesis of terminal alkenes from fermentable sugars by harnessing a P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear C10:0-C18:0 FAs, with higher activity for C16:0-C18:0 FAs. Next, we engineered a de novo alkene biosynthesis pathway, consisting of OleTMC and an engineered E. coli thioesterase (TesA) with compatible substrate specificities, and introduced this pathway into E. coli for terminal alkene biosynthesis from glucose. The recombinant E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose. To improve alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase (CamA) and a putidaredoxin (CamB) from Pseudomonas putida, into EcNN101, and demonstrated the alkene production increased ~2.8 fold. Finally, to better understand the substrate specificities of OleTMC observed, we employed in silico protein modeling to illuminate the functional role of FA binding pocket.

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

The progressive titres of key monooxygenases and their requisite native donors of reducing power were used to assess the relative contribution of various camphor plasmid (CAM plasmid)- and chromosome-coded activities to biodegradation of (rac)-camphor at successive stages throughout growth of Pseudomonas putida NCIMB 10007 on the bicylic monoterpenoid. A number of different flavin reductases (FRs) have the potential to supply reduced flavin mononucleotide to both 2,5- and 3,6-diketocamphane monooxygenase, the key isoenzymic two-component monooxygenases that delineate respectively the (+)- and (−)-camphor branches of the convergent degradation pathway. Two different constitutive chromosome-coded ferric reductases able to act as FRs can serve such as role throughout all stages of camphor-dependent growth, whereas Fred, a chromosome-coded inducible FR can only play a potentially significant role in the relatively late stages. Putidaredoxin reductase, an inducible CAM plasmid-coded flavoprotein that serves an established role as a redox intermediate for plasmid-coded cytochrome P450 monooxygenase also has the potential to serve as an important FR for both diketocamphane monooxygenases (DKCMOs) throughout most stages of camphor-dependent growth.