Studies on the selectivity of proline hydroxylases reveal new substrates including bicycles.

Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate.

Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the four-electron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of l-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.

Lysine-241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4-Catalysed Histone Demethylation.

The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2-oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC-KDM inhibitors.

Is JmjC oxygenase catalysis limited to demethylation?

Jobs on the side: Substrate selectivity studies indicate that members of the biomedically important JmjC demethylase family of histone N(ε)-methyllysine demethylases are capable of catalyzing the de-N-alkylation of groups other than N-methyl and can catalyze reactions that form stable hydroxylated products. The differences in binding preferences in this set of enzymes may be helpful in the design of selective inhibitors.

Electrical Impedance Dermography Differentiates Squamous Cell Carcinoma In Situ from Inflamed Seborrheic Keratoses.

There are no currently available low-cost, noninvasive methods for discerning the depth of squamous cell carcinoma (SCC) invasion or distinguishing SCC from its benign mimics, such as inflamed seborrheic keratosis (SK). We studied 35 subjects with subsequently confirmed SCC or SK. Subjects underwent electrical impedance dermography measurements at six frequencies to assess the electrical properties of the lesion. Averaged greatest intrasession reproducibility values were 0.630 for invasive SCC at 128 kHz, 0.444 for SCC in situ at 16 kHz, and 0.460 for SK at 128 kHz. Electrical impedance dermography modeling revealed significant differences between SCC and inflamed SK in normal skin (P < 0.001) and also between invasive SCC and SCC in situ (P < 0.001), invasive SCC and inflamed SK (P < 0.001), and SCC in situ and inflamed SK (P < 0.001). A diagnostic algorithm classified SCC in situ from inflamed SK with an accuracy of 0.958, a sensitivity of 94.6%, and a specificity of 96.9%; it also classified SCC in situ from normal skin with an accuracy of 0.796, a sensitivity of 90.2%, and a specificity of 51.2%. This study provides preliminary data and a methodology that can be used in future studies to further advance the value of electrical impedance dermography and inform biopsy decision making in patients with lesions suspicious of SCC.

Electrical Characterization of Basal Cell Carcinoma Using a Handheld Electrical Impedance Dermography Device.

Sensitive, objective, and easily applied methods for evaluating skin lesions are needed to improve diagnostic accuracy. In this study, we evaluated whether a developed noninvasive electrical impedance dermography device URSKIN could serve this purpose. In this pilot study, 17 subjects with subsequently confirmed basal cell carcinoma underwent four-electrode electrical impedance dermography measurements to assess the electrical properties of basal cell carcinoma and adjacent normal skin. A linear mixed-effects model with random intercept and slope terms was used for the analysis of multifrequency values in longitudinal and transverse directions. A significant difference in the intercept of frequency trajectories was observed for the longitudinal conductivity of 0.13 siemens/m (P < 0.001, 95% confidence interval = 0.10-0.16), transverse conductivity of 0.06 siemens/m (P < 0.001, 95% confidence interval = 0.05-0.07), longitudinal relative permittivity (dimensionless) of 203,742 (P < 0.001, 95% confidence interval = 180,292-227,191), and transverse relative permittivity (dimensionless) of 86,894 (P < 0.001, 95% confidence interval = 81,549 - 92,238). Thus, our device detected significant electrical differences between basal cell carcinoma and adjacent normal skin. Given these preliminary performance metrics and the ease of use, this technology merits further study to establish its value in facilitating the clinical diagnosis of skin cancers.

Low accuracy of self-reported family history of melanoma in high-risk patients.

Family history of melanoma is a major melanoma risk factor. However, self-reported family histories for some cancers, including melanoma, are commonly inaccurate. We used a unique database, the Utah Population Database (UPDB), as well as the Utah Cancer Registry to determine the accuracy of self-reported family history of melanoma in a large cohort of high-risk patients. Patient charts were reviewed and compared to records in the UPDB and the UCR to confirm personal and family history of melanoma in 1780 patients enrolled in a total body photography monitoring program. Self-reported family history of melanoma in first-degree relatives had an overall sensitivity of 71%, specificity of 79%, PPV of 31%, and NPV of 95%, with decreased accuracy (PPV) for second-degree relatives. A personal history of melanoma was the only factor significantly associated with accuracy in self-reported family history of melanoma. Patient age, sex, estimated nevus count, and number of prior personal melanomas were not significant predictors. Dermatologists should educate patients on the differences between melanomas, keratinocyte carcinomas, and pre-cancers. Confirming self-reported family history of melanoma may improve risk assessment for patients undergoing screening.

Improved melanoma outcomes and survival in patients monitored by total body photography: A natural experiment.

Total body photography (TBP) facilitates early melanoma detection, but long-term outcomes have not been well studied. Our objectives were to examine melanoma diagnoses, role of TBP-associated follow-up visits, and survival in patients monitored by TBP. A total of 1955 patients meeting inclusion criteria received TBP from 2004-2013 at a single academic center. We compared the melanoma diagnoses and overall survival of 1253 patients with any follow-up visits (median, three visits; range, 1-18) and 702 patients with no follow-up visits. Use of TBP photographs influenced decision to biopsy 66 of 121 (54.5%) melanomas diagnosed after TBP. Lower invasive melanoma Breslow depth was significantly associated with having one or more follow-up visit (median, 0.83 vs 0.33 mm; P = .002) and photographic review (median, 0.31 vs 0.48 mm; P = 0.02). In multivariable analyses, greater overall survival was significantly associated with having one or more follow-up visit after TBP (hazard ratio [HR], 0.36; 95% confidence interval [CI], 0.14-0.91; P < 0.032) and having more than 100 nevi (HR, 0.37; 95% CI, 0.22-0.64; P = 0.004). Worse overall survival was significantly associated with increasing age (HR per year, 1.06; 95% CI, 1.04-1.08; P < 0.001) and male sex (HR, 2.65; 95% CI, 1.48-4.73; P = 0.001). Thus, monitoring by TBP was associated with subsequent melanoma diagnoses of lower stage and depth and greater overall survival.

Studies on the Glutathione-Dependent Formaldehyde-Activating Enzyme from Paracoccus denitrificans.

Formaldehyde is a toxin and carcinogen that is both an environmental pollutant and an endogenous metabolite. Formaldehyde metabolism, which is probably essential for all aerobic cells, likely proceeds via multiple mechanisms, including via a glutathione-dependent pathway that is widely conserved in bacteria, plants and animals. However, it is unclear whether the first step in the glutathione-dependent pathway (i.e. formation of S-hydroxymethylglutathione (HMG)) is enzyme-catalysed. We report studies on glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans, which has been proposed to catalyse HMG formation from glutathione and formaldehyde on the basis of studies using NMR exchange spectroscopy (EXSY). Although we were able to replicate the EXSY results, time course experiments unexpectedly imply that GFA does not catalyse HMG formation under standard conditions. However, GFA was observed to bind glutathione using NMR and mass spectrometry. Overall, the results reveal that GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. Thus, it is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context.

Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors.

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and K(D) values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry.