Crystal Structure and Biochemical Analysis of a Cytochrome P450 CYP101D5 from Sphingomonas echinoides.

Cytochrome P450 enzymes (CYPs) are heme-containing enzymes that catalyze hydroxylation with a variety of biological molecules. Despite their diverse activity and substrates, the structures of CYPs are limited to a tertiary structure that is similar across all the enzymes. It has been presumed that CYPs overcome substrate selectivity with highly flexible loops and divergent sequences around the substrate entrance region. Here, we report the newly identified CYP101D5 from Sphingomonas echinoides. CYP101D5 catalyzes the hydroxylation of ß-ionone and flavonoids, including naringenin and apigenin, and causes the dehydrogenation of α-ionone. A structural investigation and comparison with other CYP101 families indicated that spatial constraints at the substrate-recognition site originate from the B/C loop. Furthermore, charge distribution at the substrate binding site may be important for substrate selectivity and the preference for CYP101D5.

Digital Processing for Single Nanoparticle Electrochemical Transient Measurements.

We demonstrate the use of digital frequency analysis in single nanoparticle electrochemical detection. The method uses fast Fourier transforms (FFT) of single entity electrochemical transients and digital filters. These filters effectively remove noise with the Butterworth filter preserving the amplitude of the fundamental processes in comparison with the rectangle filter. Filtering was done in three different types of experiments: single nanoparticle electrocatalytic amplification, photocatalytic amplification, and nanoimpacts of single entities. In the individual nanoparticle stepwise transients, low-pass filters maintain the step height. Furthermore, a Butterworth band-stop filter preserves the peak height in blip transients if the band-stop cutoff frequencies are compatible with the nanoparticle/electrode transient interactions. In hydrazine oxidation by single Au nanoparticles, digital filtering does not complicate the analysis of the step signal because the stepwise change of the particle-by-particle current is preserved with the rectangle, Bessel and Butterworth low pass filters, with the later minimizing time shifts. In the photocurrent single entity transients, we demonstrate resolving a step smaller than the noise. In photoelectrochemical setups, the background processes are stochastic and appear at distinct frequencies that do not necessarily correlate with the detection frequency (fp), of TiO2 nanoparticles. This lack of correlation indicates that background signals have their characteristic frequencies and that it is advantageous to perform filtering a posteriori. We also discuss selecting the filtering frequencies based on sampling rates and fp. In experiments electrolyzing ZnO, that model nanoimpacts, a band-stop filter can remove environmental noise within the sampling spectral region while preserving relevant information on the current transient. We discuss the limits of Bessel and Butterworth filters for resolving consecutive transients.

Crystal Structure and Biochemical Analysis of a Cytochrome P450 Steroid Hydroxylase (BaCYP106A6) from Bacillus Species.

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11ß-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

Insight into glucocorticoids glucosylation by glucosyltransferase: A combined experimental and in-silico approach.

Glucosyltransferases catalyze the glucosidic bond formation by transferring a glucose molecule from an activated sugar donor to an acceptor substrate. Glucocorticoids (GCs) are adrenal-derived steroid hormones most widely used for anti-inflammatory treatments. In this study, we biotransformed two selected GCs, cortisone and prednisone, into their O-glucoside derivatives using a versatile UDP-glycosyltransferase UGT-1. Complete structural assignment of glucosylated products revealed that the bioconversion by regio-selective glucosylation of cortisone and prednisone produced cortisone 21-glucoside and prednisone 21-glucoside, respectively. We also combined molecular dynamics (MD) simulation to study the binding feature and mechanism of glucosylation. MD simulation studies showed the formation of a stable complex between protein, glucose donor, and substrate, stabilized by hydrogen bonds. Overall, we were able to provide explanations for the experimentally observed selectivity for glucosylation by integrating experimental and computational techniques.

Single Entity Behavior of CdSe Quantum Dot Aggregates During Photoelectrochemical Detection.

We demonstrate that colloidal quantum dots of CdSe and CdSe/ZnS are detected during the photooxidation of MeOH, under broad spectrum illumination (250 mW/cm2). The stepwise photocurrent vs. time response corresponds to single entities adsorbing to the Pt electrode surface irreversibly. The adsorption/desorption of the QDs and the nature of the single entities is discussed. In suspensions, the QDs behave differently depending on the solvent used to suspend the materials. For MeOH, CdSe is not as stable as CdSe/ZnS under constant illumination. The photocurrent expected for single QDs is discussed. The value of the observed photocurrents, > 1 pA is due to the formation of agglomerates consistent with the collision frequency and suspension stability. The observed frequency of collisions for the stepwise photocurrents is smaller than the diffusion-limited cases expected for single QDs colliding with the electrode surface. Dynamic light scattering and scanning electron microscopy studies support the detection of aggregates. The results indicate that the ZnS layer on the CdSe/ZnS material facilitates the detection of single entities by increasing the stability of the nanomaterial. The rate of hole transfer from the QD aggregates to MeOH outcompetes the dissolution of the CdSe core under certain conditions of electron injection to the Pt electrode and in colloidal suspensions of CdSe/ZnS.

Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 from Streptomyces Species.

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

Formaldehyde Analysis in Non-Aqueous Methanol Solutions by Infrared Spectroscopy and Electrospray Ionization.

We present the analysis of formaldehyde (HCHO) in anhydrous methanol (CH3OH) as a case study to quantify HCHO in non-aqueous samples. At higher concentrations (C > 0.07 M), we detect a product of HCHO, methoxy methanol (MM, CH3OCH2OH), by Fourier transform infrared spectroscopy, FTIR. Formaldehyde reacts with CH3OH, CD3OH, and CD3OD as shown by FTIR with a characteristic spectral feature around 1,195 cm-1 for CH3OH used for the qualitative detection of MM, a formaldehyde derivative in neat methanol. Ab initio calculations support this assignment. The extinction coefficient for 1,195 cm-1 is in the order of 1.4 × 102 M-1cm-1, which makes the detection limit by FTIR in the order of 0.07 M. For lower concentrations, we performed the quantitative analysis of non-aqueous samples by derivatization with dinitrophenylhydrazine (DNPH). The derivatization uses an aqueous H2SO4 solution to yield the formaldehyde derivatized hydrazone. Ba(OH)2 removes sulfate ions from the derivatized samples and a final extraction with isobutyl acetate to yield a 1:1 methanol: isobutyl acetate solvent for injection for electrospray ionization (ESI). The ESI analysis gave a linear calibration curve for concentrations from 10 to 200 µM with a time-of-flight analyzer (TOF). The detection and quantification limits are 7.8 and 26 µM, respectively, for a linear correlation with R 2 > 0.99. We propose that the formaldehyde in CH3OH is in equilibrium with the MM species, without evidence of HCHO in solution. In the presence of water, the peaks for MM become less resolved, as expected from the well-known equilibria of HCHO that favors the formation of methylene glycol and polymeric species. Our results show that HCHO, in methanol does not exist in the aldehyde form as the main chemical species. Still, HCHO is in equilibrium between the production of MM and the formation of hydrated species in the presence of water. We demonstrate the ESI-MS analysis of HCHO from a non-aqueous TiO2 suspension in methanol. Detection of HCHO after illumination of the colloid indicates that methanol photooxidation yields formaldehyde in equilibrium with the solvent.

Characterization of CYP125A13, the First Steroid C-27 Monooxygenase from Streptomyces peucetius ATCC27952.

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated KD value, catalytic conversion rates, and Km value were 56.92 ± 11.28 µM, 1.95 nmol min-1 nmol-1, and 11.3 ± 2.8 µM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

Sinkhole susceptibility mapping in Marion County, Florida: Evaluation and comparison between analytical hierarchy process and logistic regression based approaches.

Sinkholes are the major cause of concern in Florida for their direct role on aquifer vulnerability and potential loss of lives and property. Mapping sinkhole susceptibility is critical to mitigating these consequences by adopting strategic changes to land use practices. We compared the analytical hierarchy process (AHP) based and logistic regression (LR) based approaches to map the areas prone to sinkhole activity in Marion County, Florida by using long-term sinkhole incident report dataset. For this study, the LR based model was more accurate with an area under the receiver operating characteristic (ROC) curve of 0.8 compared to 0.73 with the AHP based model. Both models performed better when an independent future sinkhole dataset was used for validation. The LR based approach showed a low presence of sinkholes in the very low susceptibility class and low absence of sinkholes in the very high susceptibility class. However, the AHP based model detected sinkhole presence by allocating more area to the high and very high susceptibility classes. For instance, areas susceptible to very high and high sinkhole incidents covered almost 43.4% of the total area under the AHP based approach, whereas the LR based approach allocated 20.7% of the total area to high and very high susceptibility classes. Of the predisposing factors studied, the LR method revealed that closeness to topographic depression was the most important factor for sinkhole susceptibility. Both models classified Ocala city, a populous city of the study area, as being very vulnerable to sinkhole hazard. Using a common test case scenario, this study discusses the applicability and potential limitations of these sinkhole susceptibility mapping approaches in central Florida.

Electrodeposition of Two-Dimensional Pt Nanostructures on Highly Oriented Pyrolytic Graphite (HOPG): The Effect of Evolved Hydrogen and Chloride Ions.

We discuss the electrodeposition of two-dimensional (2D) Pt-nanostructures on Highly Oriented Pyrolytic Graphite (HOPG) achieved under constant applied potential versus a Pt counter electrode (Eappl = ca. -2.2 V vs. NHE, normal hydrogen electrode). The deposition conditions are discussed in terms of the electrochemical behavior of the electrodeposition precursor (H2PtCl6). We performed cyclic voltammetry (CV) of the electrochemical Pt deposit on HOPG and on Pt substrates to study the relevant phenomena that affect the morphology of Pt deposition. Under conditions where the Pt deposition occurs and H2 evolution is occurring at the diffusion-limited rate (-0.3 V vs. NHE), Pt forms larger structures on the surface of HOPG, and the electrodeposition of Pt is not limited by diffusion. This indicates the need for large overpotentials to direct the 2D growth of Pt. Investigation of the possible effect of Cl- showed that Cl- deposits on the surface of Pt at low overpotentials, but strips from the surface at potentials more positive than the electrodeposition potential. The CV of Pt on HOPG is a strong function of the nature of the surface. We propose that during immersion of HOPG in the electrodeposition solution (3 mM H2PtCl6, 0.5 M NaCl, pH 2.3) Pt islands are formed spontaneously, and these islands drive the growth of the 2D nanostructures. The reducing agents for the spontaneous deposition of Pt from solution are proposed as step edges that get oxidized in the solution. We discuss the possible oxidation reactions for the edge sites.