Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family

<p>Abstract</p> <p>Background</p> <p>Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.</p> <p>Results</p> <p>The crystal structure of a putative AhpD from <it>Pseudomonas aeruginosa </it>has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from <it>Mycobacterium tuberculosis </it>despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the <it>P. aeruginosa </it>protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in <it>M. tuberculosis </it>AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of <it>P. aeruginosa </it>have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from <it>P. aeruginosa </it>exhibits a weak ability to reduce H₂O₂as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.</p> <p>Conclusion</p> <p>Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.</p

Oxygen Abundances in Two Metal-Poor Subgiants from the Analysis of the 6300 A Forbidden O I Line

Recent LTE analyses (Israelian et al. 1998 and Bosegaard et al. 1999) of the OH bands in the optical-ultraviolet spectra of nearby metal-poor subdwarfs indicate that oxygen abundances are generally higher than those previously determined. The difference increases with decreasing metallicity and reaches delta([O/Fe]) ~ +0.6 dex as [Fe/H] approaches -3.0. Employing high resolution (R = 50000), high S/N (~ 250) echelle spectra of the two stars found by Israelian et al. (1998) to have the highest [O/Fe]-ratios, viz, BD +23 3130 and BD +37 1458, we conducted abundance analyses based on about 60 Fe I and 7-9 Fe II lines. We determined from Kurucz LTE models the values of the stellar parameters, as well as abundances of Na, Ni, and the traditional alpha-elements, independent of the calibration of color vs $T_{eff}$ scales. We determined oxygen abundances from spectral synthesis of the stronger line (6300 A) of the [O I] doublet. The syntheses of the [O I] line lead to smaller values of [O/Fe], consistent with those found earlier among halo field and globular cluster giants. We obtain [O/Fe] = +0.35 +/- 0.2 for BD +23 3130 and +0.50 +/- 0.2 for BD +37 1458. In the former, the [O I] line is very weak (~ 1 mA), so that the quoted [O/Fe] value may in reality be an upper limit. Therefore in these two stars a discrepancy exists between the [O/Fe]- ratios derived from [O I] and the OH feature, and the origin of this difference remains unclear. Until the matter is clarified, we suggest it is premature to conclude that the ab initio oxygen abundances of old, metal-poor stars need to be revised drastically upward.Comment: 38 pages, 5 tables, 14 figures To appear in July 1999 AJ Updated April 16, 1999. Fixed typo

X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection

The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process

A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

Many biochemical processes take place on timescales ranging from femto­seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for in situ crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime

EDUCATIONAL SOFTWARE IN TEACHING

This paper stands as an argument for employing educational software in teaching and evaluating skills and knowledge in higher education. The authors’ main goal is to pinpoint and focus on strategies of teaching through the use of modern means, namely educational software