Native Redox Partner Research Articles

Genomic insight into O-demethylation of 4-methoxybenzoate by a two-component system from Amycolatopsis magusensis KCCM40447

Cytochrome P450 monooxygenases perform a multitude of roles, including the generation of hydroxylated aromatic compounds that might be utilized by microorganisms for their survival. WGS data of Amycolatopsis magusensis KCCM40447 revealed a complete circular genome of 9,099,986 base pairs and functionally assigned 8601 protein-encoding genes. Genomic analysis confirmed that the gene for 4-methoxybenzoate monoxygenase (CYP199A35) was conserved in close proximity to the gene for 4-hydroxybenzoate transporter (PcaK). The co-localized genes encoding CYP199A35, and ferredoxin-NAD(P) reductase (Mbr) represent a two-component system for electron transfer. CYP199A35 was specific for O-demethylation of para O-methyl substituted benzoic acid derivatives, 4-methoxybenzoate (4 MB), and 4-methoxycinnamic acid (4MCA) using the native redox partner (Mbr); two-component system and non-physiological redox partners (Pdr/Pdx); three-component system. The catalytic efficiency for O-demethylation of 4 MB using Mbr and Pdr/Pdx was 0.02 ± 0.006 min−1 μM−1 and 0.07 ± 0.02 min−1 μM−1 respectively. Further, sequence annotation and function prediction by RAST and KEEG analysis revealed a complete catabolic pathway for the utilization of 4 MB by strain KCCM40447, which was also proved experimentally.

Preparation of reductases for multicomponent oxygenases.

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.

Redox partner recognition and selectivity of cytochrome P450lin (CYP111A1)

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different.

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.

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c.

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p Ka) of the protein. However, the reduction potential of the heme iron decreases; further, the p KH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant Pseudomonas putida GS1.

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads' superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1±0.1g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55h in shake flasks and 13.3±1.9g/L in 89h in a bioreactor, the latter of which corresponds to a yield YP/S of 79%. To the authors' knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.

Redox Partner Interaction Sites in Cytochrome P450 Monooxygenases:In SilicoAnalysis and Experimental Validation

The native redox partners of many novel cytochrome P450 monooxygenases (CYPs) are unknown. Therefore, they are combined with non-native redox partners to obtain catalytically active systems. Understanding the CYP-redox partner interactions is the basis of successful protein engineering. Six redox partner interaction sites (RPISs) were identified by systematic literature, sequence, and structure analyses. All six RPISs are proposed to contribute to class II CYP-redox partner interaction interface, whereas four and five contribute to the interaction interface in class I prokaryotic and mitochondrial CYPs, respectively. The significance of the identified RPISs was tested by designing seven variants of CYP153 A (class I) as a fusion protein with its non-native redox partner CYP102 A1 reductase (class II) and measuring electron coupling efficiencies with a precision of 1–3 %. The best variant K166Q had an improved electron coupling efficiency of 64 % as compared to 53 % for the wild type.

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme.

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Understanding of real alternative redox partner of Streptomyces peucetius DoxA: Prediction and validation using in silico and in vitro analyses

Streptomyces peucetius ATCC27952 contains the cytochrome P450 monoxygenase DoxA that is responsible for the hydroxylation of daunorubicin into doxorubicin. Although S. peucetius ATCC27952 contains several potential redox partners, the most suitable endogenous electron-transport system is still unclear; therefore, we conducted a study of potential redox partners using Accelrys Discovery Studio 3.5. Recombinant DoxA along with its redox partners from S. peucetius FDX1, FDR2, and FDX3, and the putidaredoxin and putidaredoxin reductase from Pseudomonas putida that are essential equivalents of the class I type of bacterial electron-transport system were over-expressed and purified. The successful development of an efficient redox system was achieved by an in vitro enzymatic catalysis reaction with DoxA. The optimal pH for the activation of the heme was 7.6 and the optimal temperature was 30 °C. Our findings suggest a two-fold increase of DoxA activity via the NADH → FDR2 → FDX1 → DoxA pathway for the hydroxylation of the daunorubicin, and indicate that the usage of a native redox partner may increase daunorubicin-derived doxorubicin production due to the inclusion of DoxA.

Single Turnover Reveals Oxygenated Intermediates in Toluene/o-Xylene Monooxygenase in the Presence of the Native Redox Partners.

Toluene/o-xylene monooxygenase (ToMO) is a non-heme diiron protein that activates O2 for subsequent arene oxidation. ToMO utilizes four protein components, a catalytic hydroxylase, a regulatory protein, a Rieske protein, and a reductase. O2 activation and substrate hydroxylation in the presence of all four protein components is examined. These studies demonstrate the importance of native reductants by revealing reactivity unobserved when dithionite and mediators are used as the reductant. This reactivity is compared with that of other O2-activating diiron enzymes.

Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.

Protein–Protein Interaction Regulates the Direction of Catalysis and Electron Transfer in a Redox Enzyme Complex

Protein–protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein–protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis. We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein–protein interactions.

Accelerated electron transport from photosystem I to redox partners by covalently linked ferredoxin

Photosystem I is a highly efficient and potent light-induced reductase that is considered to be an appealing target for integration into hybrid solar fuel production systems. However, rapid transport of multiple electrons from the reducing end of photosystem I to downstream processes in vivo is limited by the diffusion of its native redox partner ferredoxin that is a single electron carrier. Here, we describe the design and construction of a faster electron transfer interface based on anchoring ferredoxin to the reducing end of photosystem I thereby confining the diffusion space of ferredoxin to the near vicinity of its photosystem I binding and reduction site. This was achieved by fusing ferredoxin to the PsaE subunit of photosystem I by a flexible peptide linker and reconstituting PSI in vitro with the new fusion protein. A computational algorithm was developed in order to determine the optimal linker length that will confine ferredoxin to the vicinity of photosystem I's reducing end without restricting the formation of electron transfer complexes. According to the calculation, we reconstituted photosystem I with three fusion proteins comprising PsaE and ferredoxin separated by linkers of different lengths, namely 14, 19, and 25 amino acids, and tested their effect on electron transfer rates from photosystem I to downstream processes. Indeed, we found a significant enhancement of light dependent NADPH synthesis using photosystems containing the PsaE-ferredoxin fusion proteins, equivalent to a ten-fold increase in soluble ferredoxin concentration. We propose that such a system could be used for other ferredoxin dependent redox reactions, such as the enzymatic production of hydrogen, a promising alternative fuel. As the system is comprised entirely of natural amino acids and biological cofactors, it could be integrated into the energy conversion apparatus of photosynthetic organisms by genetic engineering.

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1 fd, which is cleaved off after mitochondrial import. The physiological function of Etp1 fd is supposed to lie in the participation in the assembly of iron–sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin . Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1 fd has a melting temperature more than 20 °C higher than adrenodoxin. The three-dimensional structure of Etp1 fd has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1 fd with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1 fd and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

Electrostatic Docking of a Supramolecular Host−Guest Assembly to Cytochrome c Probed by Bidirectional Photoinduced Electron Transfer

A water-soluble octacarboxyhemicarcerand was used as a shuttle to transport redox-active substrates across the aqueous medium and deliver them to the target protein. The results show that weak multivalent interactions and conformational flexibility can be exploited to reversibly bind complex supramolecular assemblies to biological molecules. Hydrophobic electron donors and acceptors were encapsulated within the hemicarcerand, and photoinduced electron transfer (ET) between the Zn-substituted cytochrome c (MW = 12.3 kD) and the host-guest complexes (MW = 2.2 kD) was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. The behavior of the resulting ternary protein-hemicarcerand-guest assembly was investigated in two binding limits: (1) when K(encaps) ≫ K(assoc), the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium; and (2) when K(assoc) > K(encaps), the hemicarcerand-protein complex is formed first, and the hemicarcerand acts as an artificial receptor site that intercepts ligands from solution and positions them close to the active site of the metalloenzyme. In both cases, ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The measured ET rates suggest that the dominant binding region of the host-guest complex on the surface of the protein is consistent with the docking area of the native redox partner of cytochrome c. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which allow for surface-matching with the cytochrome.

Deletions in the loop surrounding the iron–sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450 scc (CYP11A1)

The redox active iron–sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe–2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe–2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450 scc (CYP11A1) indicating the importance of this loop for the physiological function of this iron–sulfur protein.

Electrocatalysis of nitrite reductase fromAlcaligenes faecalisstrain 6 mediated by native redox partner

AbstractA protein‐mediated electrocatalytic reaction is described. A blue protein fromAlcaligenes faecalisstrain 6 (AfBP) was used as a redox mediator for a nitrite reductase (NIR) from the same organism. Two steps of a homogeneous chemical reaction (AfBP‐NIR and NIR‐substrate), following the direct electron transfer reaction of AfBP (E C′C′), were analyzed, and the bimolecular rate constant of 1.8 × 106M−1·S−1for the AfBP‐NIR reaction was calculated from electrocatalytic current in cyclic voltammetry. The kinetic relation of the first and the second homogeneous reaction was discussed.

Electrochemical Behaviour of Copper-Containing Nitrite Reductase from Alcaligenes Faecalis Strain-6

Indirect and direct electrochemical reactions of copper containing nitrite reductase (NIR) from Alcaligenes faecalis strain-6 are described. The reactivity of mediators, including blue protein from the same organism (the native redox partner of NIR, AfBP), in electrocatalytic reactions (EC') involving a mediator, NIR and nitrite was investigated. Several types of EC were observed and AfBP was found to be an effective mediator in spite of its high redox potential. Direct electrochemistry was observed at an Indium Tin Oxide electrode (ITO) and an edge plane oriented pyrolytic graphite electrode (PGE). Observation of the redox activity of NIR at an ITO in an optically transparent thin layer electrode cell (OTTLE) showed that it underwent reversible changes in absorbance that corresponded to the applied potential. The electrochemically adsorbed NIR at PGE showed fast electrochemical kinetics in cyclic voltammetry. It is suggested that the weak affinity of NIR to the PGE electrode may prevent complete denatura...