The crystal structure of dihydrodipicolinate synthase from Escherichia coli with bound pyruvate and succinic acid semialdehyde: unambiguous resolution of the stereochemistry of the condensation product

The crystal structure of Escherichia coli dihydrodipicolinate synthase with pyruvate and substrate analogue succinic acid semialdehyde condensed with the active site lysine‐161 was solved to a resolution of 2.3 Å. Comparative analysis to a previously reported structure both resolves the configuration at the aldol addition center, where the final addition product clearly displays the (S)‐configuration, and the final conformation of the adduct within the active site. Direct comparison to two other crystal structures found in the Protein Data Bank, 1YXC, and 3DU0, demonstrates significant similarity between the active site residues of these structures. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Illuminating the early signaling pathway of a fungal light‐oxygen‐voltage photoreceptor

Circadian clocks are molecular timekeepers encountered in a wide variety of organisms, which allow to adapt the cell's metabolism and behavior to the daily and seasonal periods. Their function is regulated by light‐sensing proteins, among which Vivid, a light‐oxygen‐voltage (LOV) sensitive domain of the fungus Neurospora crassa, constitutes one of the most prominent examples. Although the major photochemical and structural changes during the photocycle of this photosensor have been elucidated through experimental means, its signal transduction pathway is still poorly resolved at the molecular level. In this article, we show through molecular dynamics simulation that the primary steps after adduct formation involve a switch of Gln182 in vicinity of the chromophore FAD (flavin–adenine–dinucleotide), followed by a coupling between the Iβ‐ and Hβ‐strands through H‐bond formation between Gln182 and Asn161 as well as subsequent weakening of the H‐bonding interaction between the Iβ‐ and Aβ‐strands. These processes then induce a reorientation of the Aβ‐Bβ‐loop with respect to the protein core as well as a simultaneous contraction of the partially unfolded α‐helix onto the α‐Aβ‐linker at the Ncap. Finally, we demonstrate through additional dimer simulations that the light‐induced conformational changes, observed in the monomeric case, play a decisive role in controlling the dimerization tendency of Vivid with its partner domains and that the light‐state homodimer shows a much larger affinity for aggregation than the dark state. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Absolute configurations and stability of cyclic guanosine mono-adducts with glyoxal and methylglyoxal

Glyoxal and methylglyoxal are two endogenous and mutagenic 1,2-dicarbonyl compounds, which can readily form adducts with guanosine. The molecular structures of cyclic guanosine-glyoxal (G-g) and guanosine-methylglyoxal (G-mg) mono-adducts have been extensively studied before. However, diastereoisomers of these adducts have not yet been studied in detail. In this work, one pair of G-g and two pairs of G-mg diastereoisomers were baseline separated by reverse phase HPLC, whose structures were identified as the previously reported cyclic forms, and their absolute configurations were determined by circular dichroism, the octant rule, and molecular modeling. According to the HPLC elution order, configurations of two G-g (as well as trans G-mg) were (6R,7R) and (6S,7S), respectively. Meanwhile, the stability of each isomer in neutral solution was also investigated, which revealed the stability order G-g > cis G-mg > trans G-mg and also indicated distinct transformation processes for different G-mg configurations. Trans G-mg only racemized between each other, while cis G-mg transformed to both cis and trans forms. Different intermediates in the racemization processes were proposed to explain the observations. These results may shed light on further understanding the roles of these two small molecules in mutagenesis.     

Detection of 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP)–DNA adducts in human pancreatic tissues

Recent epidemiological investigations have observed an association between the consumption of grilled or barbecued meat and an increased risk of pancreatic cancer, suggesting that dietary exposure to heterocyclic aromatic amines (HCA) may contribute to the development of this disease. 2-Amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats. To determine whether HCA-induced DNA damage is present in the human pancreas, immunohistochemistry and computer-assisted image analysis were used to measure PhIP–DNA adducts in 54 normal pancreatic tissues (N) from persons without pancreatic cancer and in 38 normal adjacent pancreatic tissues (A) and in 39 cancer tissues (T) from 68 patients with pancreatic adenocarcinoma. PhIP–DNA adducts were detected in 53 N, 34 A and 39 T samples. Mean values (±SD) of the absorbency for PhIP staining were 0.22±0.04, 0.24±0.04, and 0.24±0.03 for N, A, and T samples, respectively (p=0.004). Using the median absorbency (0.21) of the samples from normal controls as the cut-off, 71% of A and 77% of T tissues, compared with 48% of N tissues, were distributed in the higher range (p=0.009). The odds ratio of pancreatic cancer was 3.4 (95% confidence interval 1.5–7.5, p=0.002) for individuals with a higher level of PhIP–DNA adducts. This is the first report of the detection of PhIP–DNA adducts in human pancreatic tissue samples obtained from patients with unknown exposure to HCA. Although limited by the small sample size, these preliminary results suggest that PhIP exposure may contribute to human pancreatic cancer development.     

The utility of naphthyl-keratin adducts as biomarkers for jet-fuel exposure

We investigated the association between biomarkers of dermal exposure, naphthyl-keratin adducts (NKA), and urine naphthalene biomarker levels in 105 workers routinely exposed to jet-fuel. A moderate correlation was observed between NKA and urine naphthalene levels (p?=?0.061). The NKA, post-exposure breath naphthalene, and male gender were associated with an increase, while CYP2E1*6 DD and GSTT1-plus (++/+?) genotypes were associated with a decrease in urine naphthalene level (p?<?0.0001). The NKA show great promise as biomarkers for dermal exposure to naphthalene. Further studies are warranted to characterize the relationship between NKA, other exposure biomarkers, and/or biomarkers of biological effects due to naphthalene and/or PAH exposure.     

Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance

During the last three decades, 4-hydroxy-2-nonenal (HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of pathologies such as metabolic diseases, neurodegenerative diseases and cancers. These multiple pathologies can be explained by the fact that HNE is a potent modulator of numerous cell processes such as oxidative stress signaling, cell proliferation, transformation or cell death. The main objective of this review is to focus on the different aspects of HNE-induced cell death, with a particular emphasis on apoptosis. HNE is a special apoptotic inducer because of its abilities to form protein adducts and to propagate oxidative stress. It can stimulate intrinsic and extrinsic apoptotic pathways and interact with typical actors such as tumor protein 53, JNK, Fas or mitochondrial regulators. At the same time, due to its oxidant status, it can also induce some cellular defense mechanisms against oxidative stress, thus being involved in its own detoxification. These processes in turn limit the apoptotic potential of HNE. These dualities can imbalance cell fate, either toward cell death or toward survival, depending on the cell type, the metabolic state and the ability to detoxify.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Characterization of the High Resolution ESR Spectra of the Methoxyl Radical Adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)

Spin-trapping investigators are largely limited by the instability of the radical adducts. Spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms very stable alkoxyl radical adducts. However, the presence of two chiral centers in the DEPMPO alkoxyl radical adduct results in two diastereomers with distinctive ESR spectra, which complicates the interpretation of the ESR spectra. We have analyzed the high resolution ESR spectra of the DEPMPO/^?OCH³ radical adduct. DEPMPO/^?OCH³ has been synthesized by the nucleophilic addition of alcohols to DEPMPO. The electron spin resonance (ESR) spectrum of DEPMPO/^?OCH³ in oxygen-free methanol solution reveals superhyperfine structure with hyperfine coupling constants as small as 0.3?G. In order to simplify the analysis of the electron spin resonance (ESR) spectrum, we synthesized the DEPMPO/^?OCD³ radical adduct. Computer simulation of the DEPMPO/^?OCD³ ESR spectrum revealed two diastereomers. Hyperfine coupling constants of γ-protons and ¹⁷O from the –OCH³ group were also determined. ESR spectra of DEPMPO/^?OCH³ in phosphate buffer have also been characterized. The presence of specific hyperfine couplings from the –OCH³ group can be used for the unambiguous identification of the DEPMPO/^?OCH³ radical adducts. We suggest that the analysis of high resolution ESR spectra can be used for the unambiguous characterization of DEPMPO radical adducts.     

Chiral Isolation and Absolute Configuration of (+)‐ and (−)‐Xanchryones F and G from Xanthostemon chrysanthus

(+)‐ and (?)‐Xanchryones F and G ((+)‐ and (?)‐ 1 and 2 ) were isolated from the plant Xanthostemon chrysanthus by chiral separation. Compounds 1 and 2 featured a new carbon skeleton with cinnamoyltriketone‐flavone adducts. Their structures with absolute configurations were elucidated by detailed spectroscopic analyses and chemical calculations. The antibacterial and anti‐inflammatory activities of (+)‐ and (?)‐ 1 and 2 were evaluated.