Disinfect Porphyromonas gingivalis Biofilm on Titanium Surface with Combined Application of Chlorhexidine and Antimicrobial Photodynamic Therapy

Various antimicrobial modalities have been proposed to treat peri‐implantitis but resulted in limited outcomes. The aim of this in vitro study was to evaluate the disinfection efficacy of combined application of chlorhexidine digluconate (CHX) and antimicrobial photodynamic therapy (aPDT) of titanium surfaces previously contaminated with Porphyromonas gingivalis biofilm. P. gingivalis biofilms were grown on 32 polished and 32 sandblasted large‐grit acid‐etched (SLA) titanium surfaces. Titanium disks were allocated into four groups as follows: (1) immersed in phosphate‐buffered saline (PBS), (2) immersed in 0.2% CHX, (3) application of aPDT and (4) immersed in 0.2% CHX and subsequent aPDT. Residual bacteria were determined by microbial culture analysis and by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) imaging. Combination protocol (CHX+ aPDT) was the most effective in eradicating P. gingivalis (P < 0.05) on both polished and SLA surfaces. There was no significant difference in the number of remaining P. gingivalis between polished titanium disks and the SLA ones in four groups (P > 0.05). Under the limitation of this study, combined technique of preceding application of CHX and subsequent aPDT was shown to be an efficient method in reducing P. gingivalis numbers in both polished and SLA titanium surfaces.     

Preclinical Investigation of Methylene Blue-mediated Antimicrobial Photodynamic Therapy on Leishmania Parasites Using Real-Time Bioluminescence

Cutaneous leishmaniasis (CL) is a neglected disease that promotes destructive lesions. Difficulties in treatment are related to accessibility of drugs, resistance and toxicity. Antimicrobial photodynamic therapy (APDT) has been emerging as a promising treatment for CL. In this work, we evaluated methylene blue (MB)-mediated APDT (MB-APDT) on Leishmania amazonensis in vitro and in vivo by bioluminescence technique. In vitro, MB-APDT was performed using a red LED (λ = 660 ± 11 nm, 100 mW cm⁻²) and MB (100 µm ) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into three groups: control, APDT 1 (single session) and APDT 2 (two sessions of MB-APDT). MB was used at 100 µm and energy dose was established at 150 J cm⁻². Parasite burden, lesion size and pain were evaluated weekly for 4 weeks. In vitro, lethal dose for 90% parasite inactivation was achieved at 48.8 J cm⁻². In vivo, although APDT 1 and APDT 2 groups have showed similar parasite burden after 4 weeks, two sessions were clinically better, especially considering the inflammatory process associated to CL. Our findings reinforce MB-APDT as a cost-effective treatment to combat CL.     

Disinfection of Cariogenic Pathogens in Planktonic Lifestyle,Biofilm and Carious Dentine with Antimicrobial Photodynamic Therapy

Antimicrobial photodynamic therapy (aPDT) has been recommended for clinical application. Its antibacterial effect on bacteria remained in dentinal tubule was seldom investigated. Here, we evaluated the antibacterial effects of aPDT on Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) in planktonic lifestyle, biofilm and carious dentine. Mono-species biofilms or dentinal caries formed on human dentine slices or slabs. Bacterial suspension, biofilms and dentine caries were treated with 0.1 mg mL⁻¹ toluidine Blue O followed by irradiation with a light emission diode (λ − 635 ± 10 nm; 500 mW; 31.5 J cm⁻²; 60 s) and 0.12% chlorhexidine (CHX), respectively. Residual bacteria were determined by microbial culture analysis and scanning electron microscopy (SEM). One-way analysis of variance (ANOVA) was performed to detect the significance of the variables. Both treatments significantly reduced the number of L. acidophilus in planktonic state, biofilm and carious dentine (P < 0.05). For S. mutans, CHX was only bactericidal against suspension (P < 0.05), while aPDT was effective on both suspension and biofilm (P < 0.05) while not for dentin caries (P > 0.05). Under the experimental conditions assessed, aPDT could be an alternative disinfection method for superficial layer of caries cavity. Its disinfection on bacteria in dentinal tubule of deep layer was deficient.     

Antimicrobial photodynamic therapy on drug-resistant Pseudomonas aeruginosa-induced infection. An in vivo study

Pseudomonas aeruginosa is considered one of the most important pathogens that represent life‐threatening risk in nosocomial environments, mainly in patients with severe burns. Antimicrobial photodynamic therapy (aPDT) has been effective to kill bacteria. The purpose of this study was to develop a burn wound and bloodstream infection model and verify aPDT effects on it. In vitro, we tested two wavelengths (blue and red LEDs) on a clinical isolate of P. aeruginosa strain with resistance to multiple antibiotics using HB:La⁺³ as photosensitizer. Verapamil^® associated to aPDT was also studied. In vivo, P. aeruginosa‐infected burned mice were submitted to aPDT. Bacterial counting was performed on local infection and bloodstream. Survival time of animals was also monitored. In this study, aPDT was effective to reduce P. aeruginosa in vitro. In addition, Verapamil^® assay showed that HB:La⁺³ is not recognized by ATP‐binding cassete (ABC) efflux pump mechanism. In the in vivo study, aPDT was able to reduce bacterial load in burn wounds, delay bacteremia and keep the bacterial levels in blood 2–3 logs lower compared with an untreated group. Mice survival was increased on 24 h. Thus, this result suggests that aPDT may also be a novel prophylactic treatment in the care of burned patients.     

Effectiveness of the Photoactive Dye Methylene Blue versus Caspofungin on the Candida parapsilosis Biofilm in vitro and ex vivo

This research studied the effectiveness of the photoactive compound methylene blue (MB) activated with red LED light (576–672 nm) compared to that of caspofungin (CAS) on 1 Candida albicans and 3 Candida parapsilosis strains. Results were evaluated in terms of SMIC₅₀ for CAS or in PDI (photodynamic inactivation)‐SMIC₅₀ for MB (minimal inhibitory concentration inhibiting sessile biofilm to 50% in comparison to the control without CAS or after irradiation in comparison to the control without MB). While all strains were susceptible to CAS in planktonic form, the SMIC₅₀ was determined to be >16 μg mL^?1 when CAS was added to a 24 h biofilm. However, PDI‐MIC_50s (1.67 mW cm^?2, fluence 15 J cm^?2) were 0.0075–0.03 mmol L^?1. For biofilm, PDI‐SMIC_50s were in the range from 0.7 to 1.35 mmol L^?1. MB concentration of 1 mmol L^?1 prevented a biofilm being formed ex vivo on mouse tongues after irradiation regardless of the application time, in contrast to CAS, which was only effective at a concentration of 16 μg mL^?1 when it was added at the beginning of biofilm formation. PDI seems to be a promising method for the prevention of microbial biofilms that do not respond significantly to conventional drugs.     

Modified drug release system based on Sulindac and layered double hydroxide: An in vivo Raman investigation

In this study, the capability of a Layered Double Hydroxide (LDH) to promote the modified-release of Sulindac (Sul), a non-steroid anti-inflammatory drug, was followed by Raman spectroscopy through in vitro and in vivo assays. The drug and the LDH-Sul system were pressed into pellets and their dissolution under controlled conditions was monitored in vitro for 80 h. For the in vivo assays, LDH-drug and LDH-Cl (with chloride in the interlayer space) pellets were implanted between the external and internal oblique muscles of Wistar rats. The pellets were removed after 7, 21, 28 and 35 days from implantation and up to 21 days there was an increase in the amount of intercalated carbonate ions as followed over time by Raman microscopy. The carbonate ions are from the extracellular fluid (conjunctive tissue) and replace the chloride ions between the LDH layers. The same kind of ion exchange was observed for LDH-Sulindac: ca. 50% of the drug was released in vivo after 21 days, whereas in vitro the same amount of drug was delivered within 24 h. The release kinetics was much slower in the in vivo assay due to a significantly smaller hydrodynamic interaction inside the muscle.     

Hydrogen Peroxide Enhances the Antibacterial Effect of Methylene Blue‐based Photodynamic Therapy on Biofilm‐forming Bacteria

Recently, increased attention has been focused on endoscopic disinfection after outbreaks of drug‐resistant infections associated with gastrointestinal endoscopy. The aims of this study were to investigate the bactericidal efficacy of methylene blue (MB)‐based photodynamic therapy (PDT) on Pseudomonas aeruginosa (P. aeruginosa), which is the major cause of drug‐resistant postendoscopy outbreak, and to assess the synergistic effects of hydrogen peroxide addition to MB‐based PDT on biofilms. In planktonic state of P. aeruginosa, the maximum decrease was 3 log₁₀ and 5.5 log₁₀ at 20 and 30 J cm^?2, respectively, following MB‐based PDT. However, the maximum reduction of colony forming unit (CFU) was decreased by 2.5 log₁₀ and 3 log₁₀ irradiation on biofilms. The biofilm formation was significantly inhibited upon irradiation with MB‐based PDT. When the biofilm state of P. aeruginosa was treated with MB‐based PDT with hydrogen peroxide, the CFU was significantly decreased by 6 log₁₀ after 20 J cm^?2, by 7 log₁₀ after 30 J cm^?2 irradiation, suggesting significantly higher efficacy than MB‐based PDT alone. The implementation of the combination of hydrogen peroxide with MB‐based PDT through working channels might be appropriate for preventing early colonization and biofilm formation in the endoscope and postendoscopy outbreak.     

Synthesis,Nematicidal Activity,and 3D‐QSAR of Novel 1,3,4‐Oxadiazole/ Thiadiazole Thioether Derivatives

Forty one novel 1,3,4‐oxadiazole/thiadiazole thioether derivatives containing phenoxy moiety were designed and synthesized. Bioassay demonstrated that some of them showed remarkable activities against Tylenchulus semipenetrans in vitro and in vivo. Compounds 20 , 21 , 35 and 39 showed excellent lethal activities after treatment for 48 h in vitro, with LC₅₀ values of 13.4 ± 1.8, 11.7 ± 2.5, 13.7 ± 2.4 and 13.3 ± 1.1 mg·L^–1, respectively, which were obviously superior to fosthiazate (49.1 ± 2.8 mg·L^–1) and avermectin (26.6 ± 2.3 mg·L^–1). Compound 21 can effectively control the citrus nematode disease caused by T. semipenetrans at 200 mg·L^–1 in vivo with (68 ± 3)% inhibitory effect, which was even better than that of avermectin ((63 ± 2)%). The CoMFA and CoMSIA models of three‐dimensional quantitative structure‐activity relationships (3D‐QSARs) were established. The compound 33 was designed based on the 3D‐QSAR models with more vigorous nematicidal activities in vitro (LC₅₀ = 9.8 ± 1.4 mg·L^–1) and in vivo ((70 ± 5)%). These results demonstrated that compound 33 can be considered as a potential nematicide.     

Efflux Pump Inhibitor Potentiates Antimicrobial Photodynamic Inactivation of Enterococcus faecalis Biofilm

Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2-2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm-derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (P < 0.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (P < 0.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure.     

Cutaneous Photoprotective Activity of a Short‐term Ingestion of High‐Flavanol Cocoa: A Nutritional Intervention Study

In vivo and in vitro studies have shown that prolonged oral administration of flavanol‐rich cocoas extracts have photoprotective effects. The aim of the present study was to assess the photoprotective activity of short‐time administration of a new variety of naturally selected cocoa extract rich in bioactive compounds. We selected a cocoa powder particularly rich in polyphenols, flavanols, caffeine, theobromine and theophylline. We then investigated, in 10 healthy subjects, the photoprotective effects of one week of daily oral administration of two doses of such powder. Phototesting with solar simulated radiation was performed at baseline and after cocoa supplementation. Visual assessment of the minimal erythema dose and spectrophotometric measurement of the “a” parameter were done after 24 h from the irradiation. Oral daily supplementation of 1 g of high‐flavanol cocoa was not effective but a one‐week administration of 4–6 g of cocoa induced a statistically significant increase in the visual erythema threshold and a significant reduction in the “a” parameter. We demonstrated that a new variety of naturally selected cocoa extract, even when administered for a short time, retains a dose‐dependent photoprotective effect. These results are also indicative of the fact that topical sunscreens could be supplemented by a specific diet.     

Prion protein insertional mutations increase aggregation propensity but not fiber stability

Background  

Mutations in the PRNPgene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNPcause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD) expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI⁺] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI ⁺] and convert from [psi ^-] to [PSI ⁺] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivoand in vitro.     

What Caused the Formation of the Absorption Maximum at 421 nm in vivo Spectra of Rhodopseudomonas palustris

A spectral peak at ~421 nm appeared in vivo spectrum of Rhodopseudomonas palustris CQV97 cultured in acetate–glutamate medium (M1) but not in acetate–ammonium sulfate medium (M2). However, the spectral origin of 421 nm peak was not clear and frequently attributed to carotenoid component(s). In this study, comparative analysis of the extracted components showed that magnesium protoporphyrin IX monomethylester (MPE) was accumulated as one of the predominate components in M1 culture. The amounts of bacteriochlorophyll a in M1 culture were higher than that in M2, whereas the amounts of carotenoids were nearly identical in both cultures. A simple, rapid and minimum interference with carotenoid and bacteriochlorophyll method to efficiently extract the compounds involving in the formation of 421 nm peak was developed in this study. Assembly of purified MPE with protein components from R. palustris in vitro demonstrated that MPE caused the formation of 421 nm peak. The localization analysis in vivo demonstrated it is MPE associating to protein components and accounting for the peak at ~421 nm. This work clarified the 421 nm peak in vivo mainly originated from MPE accumulation, and will be very helpful to further explore the physiological roles of MPE or its derivatives in photosynthesis.     

Photodynamic Inactivation of Conidia of the Fungi Metarhizium anisopliae and Aspergillus nidulans with Methylene Blue and Toluidine Blue

Antimicrobial photodynamic treatment (PDT) is a promising method that can be used to control localized mycoses or kill fungi in the environment. A major objective of the current study was to compare the conidial photosensitization of two fungal species (Metarhizium anisopliae and Aspergillus nidulans) with methylene blue (MB) and toluidine blue (TBO) under different incubation and light conditions. Parameters examined were media, photosensitizer (PS) concentration and light source. PDT with MB and TBO resulted in an incomplete inactivation of the conidia of both fungal species. Conidial inactivation reached up to 99.7%, but none of the treatments was sufficient to achieve a 100% fungicidal effect using either MB or TBO. PDT delayed the germination of the surviving conidia. Washing the conidia to remove unbound PS before light exposure drastically reduced the photosensitization of A. nidulans. The reduction was much smaller in M. anisopliae conidia, indicating that the conidia of the two species interact differently with MB and TBO. Conidia of green and yellow M. anisopliae mutants were less affected by PDT than mutants with white and violet conidia. In contrast to what occurred in PBS, photosensitization of M. anisopliae and A. nidulans conidia was not observed when PDT was performed in potato dextrose media.     

Photoinactivation of Moraxella catarrhalis Using 405-nm Blue Light: Implications for the Treatment of Otitis Media

Moraxella catarrhalis is one of the major otopathogens of otitis media (OM) in childhood. M. catarrhalis tends to form biofilm, which contributes to the chronicity and recurrence of infections, as well as resistance to antibiotic treatment. In this study, we aimed to investigate the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative nonpharmacological approach, for the inactivation of M. catarrhalis OM. M. catarrhalis either in planktonic suspensions or 24-h old biofilms were exposed to aBL at the irradiance of 60 mW cm⁻². Under an aBL exposure of 216 J cm⁻², a >4-log₁₀ colony-forming units (CFU) reduction in planktonic suspensions and a >3-log₁₀ CFU reduction in biofilms were observed. Both transmission electron microscopy and scanning electron microscopy revealed aBL-induced morphological damage in M. catarrhalis. Ultraperformance liquid chromatography results indicated that protoporphyrin IX and coproporphyrin were the two most abundant species of endogenous photosensitizing porphyrins. No statistically significant reduction in the viability of HaCaT cells was observed after an aBL exposure of up to 216 J cm⁻². Collectively, our results suggest that aBL is potentially an effective and safe alternative therapy for OM caused by M. catarrhalis. Further in vivo studies are warranted before this optical approach can be moved to the clinics.     

Anti‐inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with Promising COX‐2 Selectivity and Enhanced Gastric Safety Profile

Novel sulfonamide containing diaryl pyrazoles were synthesized and were subsequently tested for their in vitro cyclooxygenase inhibitory assay. Compounds that showed promising in vitro COX‐2 IC₅₀ values and selectivity indices were then evaluated for their in vivo anti‐inflammatory inhibition assay using standard carrageenan‐induced rat paw edema method. Two promising inhibitors were evaluated for ulcerogenic liability. X‐ray crystal structure of COX‐2 was taken from PDB entry COX‐2 (3LN1) having a resolution of 2.80 Å (Angstroms). Structural preparations for docking studies were accomplished using protein preparation wizard in Maestro 9.0. Compound 10b displayed reasonable COX‐2 inhibition (COX‐2 IC₅₀ = 0.52 μM) and COX‐2 selectivity index (SI = 10.73) when compared with celecoxib (COX‐2 IC₅₀ = 0.78 μM) and (SI = 9.51). In vivo anti‐inflammatory studies demonstrated 64.28% inhibition for 10b in comparison with the 57.14% for that of celecoxib itself. The results of ulcerogenic liability were also found comparable with standard celecoxib. Molecular docking studies revealed that all the designed molecules showed good interactions with receptor active site with glide scores in the range −13.130 to −10.624.     

Removal of methylene blue by two zeolites prepared from naturally occurring Egyptian kaolin as cost effective technique

The optimum condition as well as adsorption behavior of two zeolite types prepared from Egyptian kaolin (namely, zeolite A and zeolite X) with methylene blue (MB) are demonstrated in this study. This will be a step to remove such dyes from textile as well as dying industries.MB removal was investigated using synthetic solutions at initial concentrations 15 mg/L of MB at constant temperature and pH (25 ± 0.1 °C and 7.5 ± 0.2) respectively. The removal efficiency was determined at different contact times and different zeolite doses.The optimum contact times for the removal of MB were 60 min and 75 min for zeolite X and zeolite A, respectively. 0.6 g was the optimum dose for removal of MB with both zeolite types.The batch method has been employed, using MB concentration in solution ranging from 2 to 25 mg/L. The percentage removal and distribution coefficients (K_d) were determined for the adsorption system as a function of sorbate concentration. The isothermal models investigated in this study show that adsorption ratios of MB on both zeolites match to Langmuir and Freundlich equation adding to that every equation constant has been calculated. According to the equilibrium studies, adsorption of zeolite X in higher concentrations is much better than that of zeolite A. Dublin–Kaganer–Radushkevich (DKR) shows physisorption endothermic adsorption process for both zeolites and also linear correlation of Redlich–Peterson and Tekman isothermal models were proved.These results show that zeolites prepared from naturally abundant Egyptian kaolin hold great potential to remove dying materials such as MB from wastewater. This will encourage using such low cost technique in removal of dyes from industrial wastewater.     

Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with ³H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.     

Pt(II) complexes immobilized on polymer‐modified magnetic carbon nanotubes as a new platinum drug delivery system

We combine nanotechnology and chemical synthesis to create a novel multifunctional platinum drug delivery vehicle based on magnetic carbon nanotubes (multiwall carbon nanotubes/Fe₃O₄@poly(citric acid)/cis‐[(Pt(1,7‐phenanthroline)(DMSO)Cl₂)]‐b‐poly(ethylene glycol) (MCNTs/FO@PC/Pt(II)‐b‐PEG)) for targeted cancer therapy. MCNTs/FO@PC/Pt(II)‐b‐PEG was conveniently prepared by conjugating cis‐[Pt(1,7‐phenanthroline)(DMSO)Cl₂] complex to MCNTs/FO@PC‐b‐PEG via strong hydrogen‐bonding interactions. In comparison with free cisplatin and Pt(II) complex, MCNTs/FO@PC/Pt(II)‐b‐PEG shows higher solubility in aqueous solution and higher cytotoxicity towards human cervical cancer HeLa cells and human breast cancer MDA‐MB‐231 cells. In vitro release experiments revealed that the platinum drug‐loaded delivery system is relatively stable under physiological conditions (pH = 7.4 and 37 °C) but susceptible to acidic environments (pH = 5.6 and 37 °C) which would trigger the release of loaded drugs. Fluorescence microscopy studies revealed that this magnetic nanohybrid system possesses marked cell‐specific targeting in vitro in the presence of an external magnetic field. The results indicated that the prepared superparamagnetic MCNTs/FO@PC/Pt(II)‐b‐PEG nanohybrid system is a promising candidate for inhibiting the proliferation of cancer cells.     

Solubilization of Hexyl Aminolevulinate by Surfactants for Tumor Fluorescence Detection

Hexyl aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinic acid (5-ALA) and can induce more protoporphyrin IX (PpIX) formation and stronger fluorescence intensity (FI) than 5-ALA, which will greatly facilitate photodynamic diagnosis and therapy. The main drawback of HAL is its low solubility in neutral aqueous media. In this study, surfactants were used to increase HAL solubility in the cell culture medium and serum, followed by in vitro fluorescence formation measurement in human pancreatic cancer cells (SW1990) and in vivo fluorescence detection in tumor-bearing mice. The results showed that Tween 80 (TW80) and Kolliphor® HS 15 (HS15) increased the solubility of HAL in the selected media. Although TW80 and HS15 exhibited in vitro cytotoxicity at high concentrations (5 mg mL⁻¹), they facilitated fluorescent signal formation at the early stage of cell incubation. When surfactants were used, the FI should be determined without the routine washing process because surfactant-containing culture medium caused the loss of synthesized PpIX during the washing process. When HAL dissolved in TW80 solution was injected intraperitoneally into pancreatic cancer-bearing mice at a dose of 50 mg kg⁻¹, the tumors exhibited red fluorescence, which indicated that systemic administration of surfactant-solubilized HAL might be applicable for tumor fluorescence detection in vivo.     

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

Background  

β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system.