Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation.

Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) variants can be visually identified via their blue phenotype. Indigo (+) single variants, indigo (-) single variants and a combinatorial library, containing mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. Recombination of indigo (+) single variants to create a multiple-variant library is a particularly useful strategy, as all top performing P450 BM3 variants with high hydroxylation activity were either indigo (+) single variants or contained multiple substitutions. Furthermore, active variants, as determined using the 4-AAP assay, were further characterized and several variants were identified that gave more than 90% conversion with 1,3-dichlorobenzene and predominantly formed 2,6-dichlorophenol; other variants showed significant substrate selectivity. This supports the hypothesis that substitution at positions that enable the indigo (+) phenotype, or hotspot residues, is a general mechanism for increasing aromatic hydroxylation activity. Overall, this research demonstrates that indigo (+) single variants, identified via colorimetric colony-based screening, may be recombined to generate a multiply-substituted variant library containing many variants with high aromatic hydroxylation activity. The combination of colony-based screening and other screening assays greatly accelerates enzyme engineering, as readily-identified indigo (+) single variants can be recombined to create a library of active multiple variants without extensive screening of single variants.

Epoxidation of perillyl alcohol by engineered bacterial cytochrome P450 BM3.

Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst. The activity of wild-type (WT) CYP102A1 and 29 engineered enzymes toward POH was screened using a high-performance liquid chromatography. They produced one major product. Among them, the engineered CYP102A1 M601 mutant with seven mutations (R47L/F81I/F87V/E143G/L150F/L188Q/E267V) showed the highest conversion, 6.4-fold higher than the WT. Structure modeling using AlphFold2 and PyMoL suggests that mutations near the water channel may be responsible for the increased catalytic activity of the M601 mutant. The major product was identified as a POH-8,9-epoxide by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. The optimal temperature and pH for the product formation were 35 °C and pH 7.4, respectively. The kcat and Km of M601 were 540 min-1 and 2.77 mM, respectively. To improve POH-8,9-epoxide production, substrate concentration and reaction time were optimized. The optimal condition for POH-8,9-epoxide production by M601 was 5.0 mM POH, pH 7.4, 35 ℃, and 6 h reaction, which produced the highest concentration of 1.72 mM. Therefore, the biosynthesis of POH-8,9-epoxide using M601 as a biocatalyst is suggested to be an efficient and sustainable synthetic process that can be applied to chemical and pharmaceutical industries.

Effect of CO binding to P450 BM3 F393 mutants on electron density distribution in the heme cofactor.

Resonance Raman spectroscopy has been performed on a set of cytochrome P450 BM3 heme domains in which mutation of the highly conserved Phe393 induces significant variation in heme iron reduction potential. In previous work [Chen, Z., Ost, T.W.B., and Schelvis, J.P.M. (2004) Biochemistry 43, 1798-1808], a correlation between heme vinyl conformation and the heme iron reduction potential indicated a steric control by the protein over the distribution of electron density in the reduced heme cofactor. The current study aims to monitor changes in electron density on the ferrous heme cofactor following CO binding. In addition, ferric-NO complexes have been studied to investigate potential changes to the proximal Cys400 thiolate. We find that binding of CO to the ferrous heme domains results in a reorientation of the vinyl groups to a largely out-of-plane conformation, the extent of which correlates with the size of the residue at position 393. We conclude that FeII dπ back bonding to the CO ligand largely takes away the need for conjugation of the vinyl groups with the porphyrin ring to accommodate FeII dπ back bonding to the porphyrin ligand. The ferrous-CO and ferric-NO data are consistent with a small decrease in σ-electron donation from the proximal Cys400 thiolate in the F393A mutant and, to a lesser extent, the F393H mutant, potentially due to a small increase in hydrogen bonding to the proximal ligand. Phe393 seems strategically placed to preserve robust σ-electron donation to the heme iron and to fine-tune its electron density by limiting vinyl group rotation.

The Dissociation Process of NADP+ from NADPH-Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation.

NADPH-cytochrome P450 reductase (CPR) plays a vital role as a redox partner for mammalian cytochrome P450 enzymes (P450s), facilitating the transfer of two electrons from NADPH to the P450 heme center in a sequential manner. Previous experimental studies revealed substantial domain movements of CPR, transitioning between closed and open states during the electron transfer (ET) cycle. These transitions are essential and are influenced by the binding of NADPH or the release of NADP+. However, the intricate molecular mechanisms governing the CPR-mediated ET cycle have largely remained elusive. This study employed molecular dynamics (MD) simulation techniques to explore the dissociation of NADP+ from CPR, a crucial step preceding the initial ET from CPR to a P450. Alongside the binding structure of NADP+ observed in a crystal structure (pose I), our MD simulations identified an alternative binding structure (pose II). Although pose II exhibits slightly lower stability than pose I, it can be formed through an approximate 210° counterclockwise rotation of the adenine group, with a free energy barrier of only 2.76 kcal/mol. The simulation results further suggest that NADP+ dissociation involves a tentative formation of pose II from pose I before complete dissociation, and that the binding of NADP+ to CPR is primarily governed by nonbonded interactions within the adenosine binding pocket.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity.

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification.

The vast majority of known enzymes exist as oligomers, which often gives them high catalytic performance but at the same time imposes constraints on structural conformations and environmental conditions. An example of an enzyme with a complex architecture is the P450 BM3 monooxygenase CYP102A1 from Bacillus megaterium. Only active as a dimer, it is highly sensitive to dilution or common immobilization techniques. In this study, we engineered a thermostable P450BM3 chimera consisting of the heme domain of a CYP102A1 variant and the reductase domain of the homologous CYP102A3. The dimerization of the hybrid was even weaker compared to the corresponding CYP102A1 variant. To create a stable dimer, we covalently coupled the C-termini of two monomers of the chimera via SpyTag003/SpyCatcher003 interaction. As a result, purification, thermostability, pH stability, and catalytic activity were improved. Via a bioorthogonal two-step affinity purification, we obtained high purity (94 %) of the dimer-stabilized variant being robust against heme depletion. Long-term stability was increased with a half-life of over 2 months at 20 °C and 80-90 % residual activity after 2 months at 5 °C. Most catalytic features were retained with even an enhancement of the overall activity by ~2-fold compared to the P450BM3 chimera without SpyTag003/SpyCatcher003.

Tryptophan extends the life of cytochrome P450.

Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450BM3) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C14) is well coupled to O2 and NADPH consumption, but shorter chains (≤C12) are more poorly coupled. Hydroxylation of p-nitrophenoxydodecanoic acid by P450BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

Computational redesign of cytochrome P450 CYP102A1 for highly stereoselective omeprazole hydroxylation by UniDesign.

Cytochrome P450 CYP102A1 is a prototypic biocatalyst that has great potential in chemical synthesis, drug discovery, and biotechnology. CYP102A1 variants engineered by directed evolution and/or rational design are capable of catalyzing the oxidation of a wide range of organic compounds. However, it is difficult to foresee the outcome of engineering CYP102A1 for a compound of interest. Here, we introduce UniDesign as a computational framework for enzyme design and engineering. We tested UniDesign by redesigning CYP102A1 for stereoselective metabolism of omeprazole (OMP), a proton pump inhibitor, starting from an active but nonstereoselective triple mutant (TM: A82F/F87V/L188Q). To shift stereoselectivity toward (R)-OMP, we computationally scanned three active site positions (75, 264, and 328) for mutations that would stabilize the binding of the transition state of (R)-OMP while destabilizing that of (S)-OMP and picked three variants, namely UD1 (TM/L75I), UD2 (TM/A264G), and UD3 (TM/A328V), for experimentation, based on computed energy scores and models. UD1, UD2, and UD3 exhibit high turnover rates of 55 ± 4.7, 84 ± 4.8, and 79 ± 5.7 min-1, respectively, for (R)-OMP hydroxylation, whereas the corresponding rates for (S)-OMP are only 2.2 ± 0.19, 6.0 ± 0.68, and 14 ± 2.8 min-1, yielding an enantiomeric excess value of 92, 87, and 70%, respectively. These results suggest the critical roles of L75I, A264G, and A328V in steering OMP in the optimal orientation for stereoselective oxidation and demonstrate the utility of UniDesign for engineering CYP102A1 to produce drug metabolites of interest. The results are discussed in the context of protein structures.

Regio- and stereo-selective 1ß-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants.

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1ß-hydroxylation of lithocholic acid (LCA) to produce 1ß-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1ß-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1ß-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp3 -H activation by the developed P450 BM3 mutants.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Investigating the applicability of the CYP102A1-decoy-molecule system to other members of the CYP102A subfamily.

Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1.

A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6-9) and temperatures (40-60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol-1 K-1. The KM values for cytochrome C and NADPH were 8.83 µM and 7.26 µM, and the kcat values were 206.79 s-1 and 202.93 s-1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min-1 mg1, the highest activity value described for a CPR up to date (3.2-4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4.

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

Biocatalytic system for comparatively assessing the functional association of monolignol cytochrome P450 monooxygenases with their redox partners.

Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3'-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b5 reductase (CBR) and/or cytochrome b5 protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant.

BACKGROUND: Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt. RESULTS: In this work, we sought to enhance BM3's total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium's own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3. CONCLUSIONS: This new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

Artificial Fusions between P450 BM3 and an Alcohol Dehydrogenase for Efficient (+)-Nootkatone Production.

Multi-enzyme cascades enable the production of valuable chemical compounds, and fusion of the enzymes that catalyze these reactions can improve the reaction outcome. In this work, P450 BM3 from Bacillus megaterium and an alcohol dehydrogenase from Sphingomonas yanoikuyae were fused to bifunctional constructs to enable cofactor regeneration and improve the in vitro two-step oxidation of (+)-valencene to (+)-nootkatone. An up to 1.5-fold increased activity of P450 BM3 was achieved with the fusion constructs compared to the individual enzyme. Conversion of (+)-valencene coupled to cofactor regeneration and performed in the presence of the solubilizing agent cyclodextrin resulted in up to 1080 mg L-1 (+)-nootkatone produced by the fusion constructs as opposed to 620 mg L-1 produced by a mixture of the separate enzymes. Thus, a two-step (+)-valencene oxidation was considerably improved through the simple method of enzyme fusion.

Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes.

Cytochrome P450 reductase (CPR) is a NADPH-dependent membrane-bound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPRox into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPRred partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the KP of CPRox insertion is measurably smaller than that of CPRred. Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane-protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.