Drug Development from Natural Products Based on the Pathogenic Mechanism of Asthma.

Asthma is a chronic inflammatory disease of the pulmonary system associated with many wheeze-to-sleep apnea complications that may lead to death. In 2019, approximately 262 million patients suffered from asthma, and 455 thousand died from the disease worldwide. It is a more severe health problem in children and older adults, and as the aging of society intensifies, the problem will continue to worsen. Asthma inducers can be classified as indoor and outdoor allergens and can cause asthma due to their repeated invasion. There are several theories about asthma occurrence, such as the imbalance between Th1 and Th2, inflammation in the pulmonary system, and the abnormal apoptosis/cell proliferation of cells related to asthma. Although there are many medications for asthma, as it is an incurable disease, the purpose of the drugs is only to suppress the symptoms. The current drugs can be divided into relievers and controllers; however, as they have many adverse effects, such as immune suppression, growth retardation, promotion of cataracts, hyperactivity, and convulsions, developing new asthma drugs is necessary. Although natural products can have adverse effects, the development of asthma drugs from natural products may be beneficial, as some have anti-asthmatic effects such as immune modulation, anti-inflammation, and/or apoptosis modulation.

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

OBJECTIVES: To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. RESULTS: CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CONCLUSIONS: CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Transformation of Escherichia coli and protein expression using lipoplex mimicry.

We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step.

Cargo-Free Nanoparticles Containing Cationic Lipids Induce Reactive Oxygen Species and Cell Death in HepG2 Cells.

Nanoparticles (NPs) containing cationic monovalent lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA), have been widely used for the delivery of nucleic acid such as small-interfering RNA and polypeptide to cells as cancer therapies and vaccine development. Several previous reports have suggested that cationic liposomes induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. Here, we systematically investigated the effects of DOTAP- or DOTMA-containing NPs without any cargo on the human carcinoma cells, HepG2. Treatment with NPs containing DOTAP or DOTMA increased the production of cellular ROS, such as H2O2 and lipid peroxidation, in HepG2 cells and concomitantly decreased cell viability. These effects were dependent on the lipid concentration, surface density of cationic lipids, and particle size of NPs. However, neutral NPs consisting of 1,2-dioleoyl-3-phosphocholine did not elicit the effective ROS generation or cell death regardless of the lipid concentration and particle size. The present study suggests that DOTAP- and DOTMA-NPs are able to induce cancer cell death through production of ROS in the absence of any therapeutic cancer reagents. These results also provide a rational background for the design of delivery systems using cationic lipid-based NP formulations.

Regioselective hydroxylation of omeprazole enantiomers by bacterial CYP102A1 mutants.

A large set of Bacillus megaterium CYP102A1 mutants are known to metabolize various drugs to form human metabolites. Omeprazole (OMP), a proton pump inhibitor, has been widely used as an acid inhibitory agent for the treatment of gastric acid hypersecretion disorders. It is primarily metabolized by human CYP2C19 and CYP3A4 to 5'-OH OMP and a sulfone product, respectively. It was recently reported that several CYP102A1 mutants can oxidize racemic and S-OMP to 5'-OH OMP and that these mutants can further oxidize 5'-OH racemic OMP to 5'-COOH OMP. Here, we report that the S- and R-enantiomers of OMP are hydroxylated by 26 mutants of CYP102A1 to produce 1 major metabolite (5'-OH OMP) regardless of the chirality of the parent substrates. Although the binding of R-OMP to the CYP102A1 active site caused a more apparent change of heme environment compared with binding of S-OMP, there was no correlation between the spectral change upon substrate binding and catalytic activity of either enantiomer. The 5'-OH OMP produced from racemic, S-, and R-OMP could be obtained with a high conversion rate and high selectivity when the triple R47L/F87V/L188Q mutant was used. These results suggest that bacterial CYP102A1 mutants can be used to produce the human metabolite 5'-OH OMP from both the S- and R-enantiomers of OMP.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.

Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems.

Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipids. This method does not require any protein-tagging system for protein isolation and has a further advantage that the purification is concomitantly conducted with reconstitution of the enzymes into a phospholipid environment, which is crucial for the catalytic activity assay of P450 enzyme. This method may also be applied to high-throughput catalytic assays of the enzymes because the purification procedures can be undertaken in a 96-well plate.

Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 µM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

Regioselective hydroxylation of 17ß-estradiol by mutants of CYP102A1 from Bacillus megaterium.

A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17ß-estradiol (E2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E2 in this study. Some of the tested mutants could regioselectively generate 2-OH E2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.

Therapeutic treatments for diabetes mellitus-induced liver injury by regulating oxidative stress and inflammation.

Diabetes mellitus (DM) is a metabolic disease that affects all systems in the body, including the liver. Numerous studies have reported that chronic DM etiology and pathogenesis complications implicate oxidative stress, generating reactive oxygen species, such as superoxide anions and free radicals. In addition, pro-inflammatory reactions are also underlying functions closely related to oxidative stress that further exacerbate pathological DM states. The liver is especially susceptible to hyperglycemia-induced oxidative stress and the related inflammation. Thus, anti-oxidation and anti-inflammation therapies are promising strategies for treating liver damage. This review summarizes therapeutic treatments attenuating the generation of oxidative stress and pro-inflammation, which also cause DM-induced liver injury. Although the treatments have several impediments to be solved, these remedies may have clinically important implications under the absence of effective drugs for the damaged liver in DM patients.

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase).

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.

Effects of phospholipids on the functional regulation of tBID in membranes.

The functional interplay between tBID and phospholipids was investigated in this study. The binding of tBID to model membranes was increased by an incorporation of phosphatidylserine (PS) into the liposomes. Using limited proteolysis and mass spectrometry, two peptide regions, which correspond to Ser(100)-Arg(114) and His(89)-Arg(114) in BID, revealed the specific PS-binding site. tBID also decreased the light scattering values of PS-containing liposomes and increased the leakage of fluorescent dye encapsulated in vesicles, which suggest that tBID reduces membrane integrity by fragmentation. The membrane fragmentation by tBID was also observed using confocal and transmission electron microscopy. The activity of tBID paralleled results that were obtained with cardiolipin (CL)-containing membranes. However, other anionic phospholipids had little effect. CL- and PS-induced conformational changes of tBID were observed by circular dichroism and intrinsic fluorescence. CL and PS also stimulated the insertion of BID into lipid monolayers. tBID stimulated the leakage of Ca(2+) from purified microsomes and mitochondria in a protein concentration-dependent manner. In contrast, BID showed significantly reduced effects when compared to tBID in all of the experiments performed. These results suggest that tBID specifically interacts with PS as well as CL and decreases membrane integrity without the aid of other pro-apoptotic proteins.

Chlorogenic acid attenuates pro-inflammatory response in the blood of streptozotocin-induced diabetic rats.

BACKGROUND: Chlorogenic acid (CGA) has been shown to reduce pro-inflammation by scavenging reactive oxygen species (ROS) and reactive nitrogen species. In this study, the anti-inflammatory effect of CGA was expanded to streptozotocin (STZ)-induced diabetic rats. The inter-relationships among oxidative stress, pro-inflammation, and cytochrome P450 (CYP) 1A enzymes were also investigated in peripheral blood mononuclear cells (PBMC) of STZ-diabetic rats. RESULTS: The levels of pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor-alpha, increased by approximately 3.4- and 2.9-fold, respectively, and the albumin concentration decreased in the serum of STZ-induced diabetic rats compared to normal rats. The C-reactive protein (CRP) values also increased by about 3.8-fold higher, indicating that STZ induced an inflammation in the blood of STZ-diabetic rats. The expression levels and catalytic activities of CYP1A enzymes were elevated by approximately 2.2-2.5- and 4.3-6.7-fold, respectively, in the PBMC of STZ-treated rats. A decrease in the amount of PBMC-bound albumin was also observed. In contrast, the levels of cytokines and CRP in serum and the activities of CYP1A enzymes in PBMC were significantly reduced in CGA-treated diabetic rats in a CGA concentration-dependent manner. In addition, STZ-mediated elevation of ROS in serum and PBMC was decreased by the CGA administration. However, the CGA treatment did not change the enhanced blood glucose level and expression of CYP1A enzymes by STZ. STZ-mediated decrease in the levels of serum and PBMC-bound albumin was not also restored by the CGA administration. CONCLUSIONS: These results suggest that CGA could be used to treat type 1 diabetes-induced inflammation.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

Multiple Porous Synthetic Bone Graft Comprising EngineeredMicro-Channel for Drug Carrier and Bone Regeneration.

Due to high demand but limited supply, there has been an increase in the need to replace autologous bone grafts with alternatives that fulfill osteogenic requirements. In this study, two different types of bone grafts were tested for their drug carrying abilities along with their osteogenic properties. Two different types of alendronate-loaded bone grafts, Bio-Oss (bovine bone graft) and InRoad (biphasic synthetic bone graft) were observed to see how different concentrations of alendronate would affect the sustained release to enhance osteogenesis. In this study, defected ovariectomize-induced osteoporotic rat calvarias were observed for 28 days with three different concentrations of alendronate (0 mg, 1 mg, 5 mg) for both Bio-Oss and InRoad. A higher concentration (5 mg) allowed for a more controlled and sustained release throughout the 28-day comparison to those of lower concentrations (0 mg, 1 mg). When comparing Bio-Oss and InRoad through histology and Micro-CT, InRoad showed higher enhancement in osteogenesis. Through this study, it was observed that alendronate not only brings out robust osteogenesis with InRoad bone grafts, but also enhances bone regeneration in an alendronate-concentration-dependent manner. The combination of higher concentration of alendronate and multiple porous bone graft containing internal micro-channel structure of InRoad resulted in higher osteogenesis with a sustained release of alendronate.

Respiratory and Systemic Toxicity of Inhaled Artificial Asian Sand Dust in Pigs.

Air pollution, particularly caused by Asian sand dust (ASD) and particulate matter (PM), has become one of the leading threats to public health. However, the majority of studies have primarily focused on epidemiological assessment, and in vivo toxicities of certain air pollutants have been poorly elucidated in medium/large-size laboratory animals. To investigate the impact of ASD in domestic animals, 16 Landrace pigs were exposed to an artificial ASD sandstorm for 6 h. All animals were divided in four cages, and a commercial yellow soil was used for generating artificial mineralogical particles. Blood samples were collected, and necropsies were performed before exposure and 6, 12, 24, and 72 h after exposure. Complete blood cell count and the levels of serum biochemical enzymes, blood gas, electrolytes, and a variety of inflammatory cytokines were evaluated. In addition, histopathological examination was conducted. Various test results proved acute lower airway disorders with systemic inflammation in pigs. To our knowledge, this study is the first to describe experimental research in domestic animals concerning the damage caused by artificial ASD exposure. The results of this study suggest that ASD has importance in terms of not only public health but also of ultimate economic losses in the pork industry.

Korean Red Ginseng affects ovalbumin-induced asthma by modulating IL-12, IL-4, and IL-6 levels and the NF-κB/COX-2 and PGE2 pathways.

BACKGROUND: Asthma is an incurable hyper-responsive disease of the pulmonary system that is caused by various allergens, including indoor and outdoor stimulators. According to the Global Asthma Network, 339 million people suffered from asthma in 2018, with particularly severe forms in children. Numerous treatments for asthma are available; however, they are frequently associated with adverse effects such as growth retardation, neurological disorders (e.g., catatonia, poor concentration, and insomnia), and physiological disorders (e.g., immunosuppression, hypertension, hyperglycemia, and osteoporosis). METHODS: Korean Red Ginseng has long been used to treat numerous diseases in many countries, and we investigated the anti-asthmatic effects and mechanisms of action of Korean Red Ginseng. Eighty-four BALB/c mice were assigned to 6 treatment groups: control, ovalbumin-induced asthma group, dexamethasone treatment group, and 3 groups treated with Korean Red Ginseng water extract (KRGWE) at 5, 25, or 50 mg/kg/day for 5 days. Anti-asthmatic effects of KRGWE were assessed based on biological changes, such as white blood cell counts and differential counts in the bronchoalveolar lavage fluid, serum IgE levels, and histopathological changes in the lungs, and by examining anti-asthmatic mechanisms, such as the cytokines associated with Th1, Th2, and Treg cells and inflammation pathways. RESULTS: KRGWE affected ovalbumin-induced changes, such as increased white blood cell counts, increased IgE levels, and morphological changes (mucous hypersecretion, epithelial cell hyperplasia, inflammatory cell infiltration) by downregulating cytokines such as IL-12, IL-4, and IL-6 via GATA-3 inactivation and suppression of inflammation via NF-κB/COX-2 and PGE2 pathways. CONCLUSION: KRGWE is a promising drug for asthma treatment.

Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation.

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.