A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.

Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.

Electrocatalytic Aldehyde Oxidation by a Tungsten Dependent Aldehyde Oxidoreductase from Aromatoleum Aromaticum.

In contrast to their molybdenum dependent relatives, tungsten enzymes operate at significantly lower redox potentials, and in some cases they can carry out reversible redox transformations of their substrates and products. Still, the electrochemical properties of W enzymes have received much less attention than their Mo relatives. Herein we analyse the tungsten enzyme aldehyde oxidoreductase (AOR) from the mesophilic bacterium Aromatoleum aromaticum which has been immobilised on a glassy carbon working electrode. This generates a functional system that electrochemically oxidises a wide variety of aromatic and aliphatic aldehydes in the presence of the electron transfer mediators benzyl viologen, methylene blue or dichlorophenol indophenol. Simulation of the cyclic voltammetry has enabled a thorough kinetic analysis of the system, which reveals that methylene blue acts as a two-electron acceptor. In contrast, the other two mediators act as single electron oxidants. The different electrochemical driving forces imparted by these mediators also lead to significantly different outer sphere electron transfer rates with AOR. This work shows that electrocatalytic aldehyde oxidation can be achieved at a low applied electrochemical potential leading to an extremely energy efficient catalytic process.

Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans─The Role of a New Putative Membrane-Associated Domain and Proton-Relay System in Catalysis.

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.

Biotransformation of Δ1-Progesterone Using Selected Entomopathogenic Filamentous Fungi and Prediction of Its Products' Bioactivity.

This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δ1-progesterone) (2) through microbiological transformation. For the role of catalysts, we used six strains of entomopathogenic filamentous fungi (Beauveria bassiana KCh J1.5, Beauveria caledonica KCh J3.3, Isaria fumosorosea KCh J2, Isaria farinosa KCh KW1.1, Isaria tenuipes MU35, and Metarhizium robertsii MU4). The substrate (2) was obtained by carrying out an enzymatic 1,2-dehydrogenation on an increased scale (3.5 g/L) using a recombinant cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans. All selected strains were characterized by the high biotransformation capacity for the used substrate. As a result of the biotransformation, six steroid derivatives were obtained: 11α-hydroxypregn-1,4-diene-3,20-dione (3), 6ß,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6ß-hydroxypregn-1,4-diene-3,11,20-trione (5), 6ß,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6ß,17ß-dihydroxyandrost-1,4-diene-3-one (7), and 12ß,17α-dihydroxypregn-1,4-diene-3,20-dione (8). The results show evident variability of the biotransformation process between strains of the tested biocatalysts from different species described as entomopathogenic filamentous fungi. The obtained products were tested in silico using cheminformatics tools for their pharmacokinetic and pharmacodynamic properties, proving their potentially high biological activities. This study showed that the obtained compounds may have applications as effective inhibitors of testosterone 17ß-dehydrogenase. Most of the obtained products should, also with a high probability, find potential uses as androgen antagonists, a prostate as well as menopausal disorders treatment. They should also demonstrate immunosuppressive, erythropoiesis-stimulating, and anti-inflammatory properties.

1,2-Hydrogenation and Transhydrogenation Catalyzed by 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans-Kinetics, Isotope Labelling and QM:MM Modelling Studies.

Bacteria and fungi that are able to metabolize steroids express 3-ketosteroid-Δ1-dehydrogenases (KstDs). KstDs such as AcmB form Sterolibacterium denitrificans Chol-1 catalyze the enantioselective 1α,2ß-dehydrogenation of steroids to their desaturated analogues, e.g., the formation of 1,4-androstadiene-3,17-dione (ADD) from 4-androsten-3,17-dione (AD). The reaction catalyzed by KstD can be reversed if the appropriate electron donor, such as benzyl viologen radical cation, is present. Furthermore, KstDs can also catalyze transhydrogenation, which is the transfer of H atoms between 3-ketosteroids and 1-dehydrosteroids. In this paper, we showed that AcmB exhibits lower pH optima for hydrogenation and dehydrogenation by 3.5-4 pH units than those observed for KstD from Nocardia corallina. We confirmed the enantiospecificity of 1α,2ß-hydrogenation and 1α,2ß-transhydrogenation catalyzed by AcmB and showed that, under acidic pH conditions, deuterons are introduced not only at 2ß but also at the 1α position. We observed a higher degree of H/D exchange at Y363, which activates the C2-H bond, compared to that at FAD, which is responsible for redox at the C1 position. Furthermore, for the first time, we observed the introduction of the third deuteron into the steroid core. This effect was explained through a combination of LC-MS experiments and QM:MM modelling, and we attribute it to a decrease in the enantioselectivity of C2-H activation upon the deuteration of the 2ß position. The increase in the activation barrier resulting from isotopic substitution increases the chance of the formation of d3-substituted 3-ketosteroids. Finally, we demonstrate a method for the synthesis of 3-ketosteroids chirally deuterated at 1α,2ß positions, obtaining 1α,2ß-d2-4-androsten-3,17-dione with a 51% yield (8.61 mg).

Biocidal Activity of Tannic Acid-Prepared Silver Nanoparticles towards Pathogens Isolated from Patients with Exacerbations of Chronic Rhinosinusitis.

The microbiome's significance in chronic rhinosinusitis (CRS) is unclear. Antimicrobials are recommended in acute exacerbations of the disease (AECRS). Increasing rates of antibiotic resistance have stimulated research on alternative therapeutic options, including silver nanoparticles (AgNPs). However, there are concerns regarding the safety of silver administration. The aim of this study was to assess the biological activity of tannic acid-prepared AgNPs (TA-AgNPs) towards sinonasal pathogens and nasal epithelial cells (HNEpC). The minimal inhibitory concentration (MIC) for pathogens isolated from patients with AECRS was approximated using the well diffusion method. The cytotoxicity of TA-AgNPswas evaluated using an MTT assay and trypan blue exclusion. A total of 48 clinical isolates and 4 reference strains were included in the study (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Klebsiellaoxytoca, Acinetobacter baumannii, Serratia marcescens, Enterobacter cloacae). The results of the studies revealed that the MIC values differed between isolates, even within the same species. All the isolates were sensitive to TA-AgNPs in concentrations non-toxic to human cells during 24 h exposition. However, 48 h exposure to TA-AgNPs increased toxicity to HNEpC, narrowing their therapeutic window and enabling 19% of pathogens to resist the TA-AgNPs' biocidal action. It was concluded that TA-AgNPs are non-toxic for the investigated eukaryotic cells after short-term exposure and effective against most pathogens isolated from patients with AECRS, but sensitivity testing may be necessary before application.

Tungsten Enzyme Using Hydrogen as an Electron Donor to Reduce Carboxylic Acids and NAD.

Tungsten-dependent aldehyde oxidoreductases (AORs) catalyze the oxidation of aldehydes to acids and are the only known enzymes reducing non-activated acids using electron donors with low redox potentials. We report here that AOR from Aromatoleum aromaticum (AOR Aa ) catalyzes the reduction of organic acids not only with low-potential Eu(II) or Ti(III) complexes but also with H2 as an electron donor. Additionally, AOR Aa catalyzes the H2-dependent reduction of NAD+ or benzyl viologen. The rate of H2-dependent NAD+ reduction equals to 10% of that of aldehyde oxidation, representing the highest H2 turnover rate observed among the Mo/W enzymes. As AOR Aa simultaneously catalyzes the reduction of acids and NAD+, we designed a cascade reaction utilizing a NAD(P)H-dependent alcohol dehydrogenase to reduce organic acids to the corresponding alcohols with H2 as the only reductant. The newly discovered W-hydrogenase side activity of AOR Aa may find applications in either NADH recycling or conversion of carboxylic acids to more useful biochemicals.

Model Setup and Procedures for Prediction of Enzyme Reaction Kinetics with QM-Only and QM:MM Approaches.

The enzyme-catalyzed reactions are traditionally studied with experimental kinetic assays. The modern theoretical modeling techniques provide a complementary way to investigate these catalytic reactions. Experimental assay frequently does not allow an unequivocal answer to the factors controlling the reaction mechanism. On the other hand, the theoretical experiments provide a precise understanding of the molecular-level steps involved in catalytic reactions. However, modeling requires at least structural data on the enzyme and reactant, and the complexity of the enzyme systems can still be a challenge.In this chapter, we are going to describe how to apply theoretical modeling methods, such as MD simulation, QM-only cluster models of enzyme active site, or QM:MM multiscale modeling to study enzyme kinetics and even to predict kinetic isotope effect (KIE). We present a full protocol that starts from the PDB structure of the enzyme, through MD simulation of enzyme: substrate complex and statistical analysis of MD trajectory, selection of a model of the active site, and study of reaction pathways. We show how theoretical predictions basing on QM-only cluster models, QM:MM model, or multiple QM:MM models derived from QM:MM:MD simulations can be correlated with experimental kinetic results. Finally, we show how one can calculate intrinsic KIE associated with an individual molecular step.

Antibacterial and Antifungal Properties of Silver Nanoparticles-Effect of a Surface-Stabilizing Agent.

The biocidal properties of silver nanoparticles (AgNPs) prepared with the use of biologically active compounds seem to be especially significant for biological and medical application. Therefore, the aim of this research was to determine and compare the antibacterial and fungicidal properties of fifteen types of AgNPs. The main hypothesis was that the biological activity of AgNPs characterized by comparable size distributions, shapes, and ion release profiles is dependent on the properties of stabilizing agent molecules adsorbed on their surfaces. Escherichia coli and Staphylococcus aureus were selected as models of two types of bacterial cells. Candida albicans was selected for the research as a representative type of eukaryotic microorganism. The conducted studies reveal that larger AgNPs can be more biocidal than smaller ones. It was found that positively charged arginine-stabilized AgNPs (ARGSBAgNPs) were the most biocidal among all studied nanoparticles. The strongest fungicidal properties were detected for negatively charged EGCGAgNPs obtained using (-)-epigallocatechin gallate (EGCG). It was concluded that, by applying a specific stabilizing agent, one can tune the selectivity of AgNP toxicity towards desired pathogens. It was established that E. coli was more sensitive to AgNP exposure than S. aureus regardless of AgNP size and surface properties.

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids.

BACKGROUND: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-ß-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. RESULTS: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 µM) than to AD (KmA = 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M-1 s-1). CONCLUSIONS: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Challenging the gold standard: methods of sampling for microbial culture in patients with chronic rhinosinusitis.

PURPOSE: Chronic rhinosinusitis (CRS) is a highly prevalent multifactorial disorder. Culture-directed antibiotics are frequently prescribed to patients with CRS and the middle nasal meatus (MM) is traditionally believed to be a representative sampling site of the sinuses as a whole. The purpose of our study was to reevaluate the reliability of the MM as a sampling site in patients with CRS who suffer from impaired drainage from the sinuses to the MM. METHODS: Swabs and tissue biopsies were collected from the MM, maxillary sinus and frontal sinus from 50 patients with CRS. The results of bacterial culture were compared between sampling methods and sites in relation to the patency of the sinus ostia. RESULTS: 782 bacterial isolates were cultured from the samples. Concordant results between the MM and the sinus cavity were noted in 80% of patients for the maxillary sinus, but only 66% for the frontal sinus and 76% for the sinuses a whole. The differences were similarly prevalent in patients with open and occluded sinus ostia. Notably, swabs from all three sites provided representative information in 92% of patients and tissue biopsies did not provide additional information compared to multiple swabs. CONCLUSION: The traditional method of sampling from the middle meatus provides inadequate information in 24% of patients with CRS, which may result in inadequate antibiotic therapy and contribute to increasing antibiotic resistance. Additional sampling from the sinuses should be recommended whenever possible, while invasive sampling is not necessary.

The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol - DCPIP, phenazine methosulfate - PMS or potassium hexacyanoferrate - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4·105 s-1·M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0·105 s-1·M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5·102 s-1·M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20-C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost-4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-ß-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 µM of isolated enzyme, PMS and aerobic atmosphere resulting in >99% conversion of the C17-C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.

Exacerbations of Chronic Rhinosinusitis-Microbiology and Perspectives of Phage Therapy.

The chronically inflamed mucosa in patients with chronic rhinosinusitis (CRS) can additionally be infected by bacteria, which results in an acute exacerbation of the disease (AECRS). Currently, AECRS is universally treated with antibiotics following the guidelines for acute bacterial rhinosinusitis (ABRS), as our understanding of its microbiology is insufficient to establish specific treatment recommendations. Unfortunately, antibiotics frequently fail to control the symptoms of AECRS due to biofilm formation, disruption of the natural microbiota, and arising antibiotic resistance. These issues can potentially be addressed by phage therapy. In this study, the endoscopically-guided cultures were postoperatively obtained from 50 patients in order to explore the microbiology of AECRS, evaluate options for antibiotic treatment, and, most importantly, assess a possibility of efficient phage therapy. Staphylococcus aureus and coagulase-negative staphylococci were the most frequently isolated bacteria, followed by Haemophilus influenzae, Pseudomonas aeruginosa, and Enterobacteriaceae. Alarmingly, mechanisms of antibiotic resistance were detected in the isolates from 46% of the patients. Bacteria not sensitive to amoxicillin were carried by 28% of the patients. The lowest rates of resistance were noted for fluoroquinolones and aminoglycosides. Fortunately, 60% of the patients carried bacterial strains that were sensitive to bacteriophages from the Biophage Pharma collection and 81% of the antibiotic-resistant strains turned out to be sensitive to bacteriophages. The results showed that microbiology of AECRS is distinct from ABRS and amoxicillin should not be the antibiotic of first choice. Currently available bacteriophages could be used instead of antibiotics or as an adjunct to antibiotics in the majority of patients with AECRS.

New lupeol esters as active substances in the treatment of skin damage.

The purpose of the research was to obtain new derivatives of natural triterpene lupeol and to evaluate their potential as active substances in the treatment of skin damage. Four new lupeol esters (propionate, succinate, isonicotinate and acetylsalicylate) and lupeol acetate were obtained using an eco-friendly synthesis method. In the esterification process, the commonly used hazardous reagents in this type of synthesis were replaced by safe ones. This unconventional, eco-friendly, method is particularly important because the compounds obtained are potentially active substances in skin care formulations. Even trace amounts of hazardous reagents can have a toxic effect on damaged or irritated tissues. The molecular structure of the esters were confirmed by 1H NMR, 13C NMR and IR spectroscopy methods. Their crystal structures were determined using XRD method. To complete the analysis of their characteristics, physicochemical properties (melting point, lipophilicity, water solubility) and biological activity of the lupeol derivatives were studied. Results of an irritant potential test, carried out on Reconstructed Human Epidermis (RHE), confirmed that the synthesized lupeol derivatives are not cytotoxic and they stimulate a process of human cell proliferation. The safety of use for tested compounds was determined in a cell viability test (cytotoxicity detection kit based on the measurement of lactate dehydrogenase activity) for keratinocytes and fibroblasts. The results obtained showed that the modification of lupeol structure improve its bioavailability and activity. All of the esters penetrate the stratum corneum and the upper layers of the dermis better than the maternal lupeol. Lupeol isonicotinate, acetate and propionate were the most effective compounds in a stimulation of the human skin cell proliferation process. This combination resulted in an increase in the concentration of cells of more than 30% in comparison to control samples. The results indicate that the chemical modification of lupeol allows to obtain promising active substances for treatment of skin damage, including thermal, chemical and radiation burns.

Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques.

In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme's specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme's isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 µg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.

Correction to: Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms.

The published online version contains mistake in the author list. The correct presentation should have been "Rita Bernhardt" instead of "Rita Bernhard". There was a missing "t" on the original publication.

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms.

The steroid superfamily includes a wide range of compounds that are essential for living organisms of the animal and plant kingdoms. Structural modifications of steroids highly affect their biological activity. In this review, we focus on hydroxylation of steroids by bacterial hydroxylases, which take part in steroid catabolic pathways and play an important role in steroid degradation. We compare three distinct classes of metalloenzymes responsible for aerobic or anaerobic hydroxylation of steroids, namely: cytochrome P450, Rieske-type monooxygenase 3-ketosteroid 9α-hydroxylase, and molybdenum-containing steroid C25 dehydrogenases. We analyze the available literature data on reactivity, regioselectivity, and potential application of these enzymes in organic synthesis of hydroxysteroids. Moreover, we describe mechanistic hypotheses proposed for all three classes of enzymes along with experimental and theoretical evidences, which have provided grounds for their formulation. In case of the 3-ketosteroid 9α-hydroxylase, such a mechanistic hypothesis is formulated for the first time in the literature based on studies conducted for other Rieske monooxygenases. Finally, we provide comparative analysis of similarities and differences in the reaction mechanisms utilized by bacterial steroid hydroxylases.

Electrocatalytic Hydroxylation of Sterols by Steroid C25 Dehydrogenase from Sterolibacterium denitrificans.

The electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the ß-Proteobacterium Sterolibacterium denitrificans is reported. S25DH catalyses the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxycholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3 ) is also a substrate. S25DH was immobilised on a modified gold working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6 ]3- ) and ferrocenium methanol (FM+ ) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-ß-cyclodextrin (HPCD) was employed as a sterol solubiliser, in addition to 2-methoxyethanol. The catalytic activity varied, depending upon the concentration of solubiliser in the reaction mixture. Parallel studies with [Fe(CN)6 ]3- as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer, pre-vitD3 , and that the former substrate is stabilised by HPCD.

Can phage therapy solve the problem of recalcitrant chronic rhinosinusitis?

Chronic rhinosinusitis (CRS) affects 5-15% of the global population. In some patients, the infectious exacerbations of the disease are recalcitrant to medical treatment and surgery. These cases are probably associated with the presence of bacterial biofilms. Bacteriophage (phage) therapy seems to be a promising antibiofilm strategy. The efficacy of phage therapy in sinonasal infections has been demonstrated both in vitro and in animal models. In the past, phage preparations were also administered to humans with CRS with favorable outcomes and no significant side effects. Very recently, the safety and efficacy of phage therapy in otolaryngological infections has been demonstrated in pioneer Phase I/II clinical trials. This review addresses the potential of phage therapy to treat CRS. We also discuss issues that require further research.

Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O2-independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[2H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH.