Photoreduction of the Folate Cofactor in Members of the Photolyase Family

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH⁻* to the DNA lesion and electron back-transfer to semireduced FADH^o after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.DNA photolyases and cryptochromes (cry)² form a large family of related flavoproteins with DNA repair activity and photoreceptor function, respectively. Members of this protein family were identified in all kingdoms of life and can be grouped in at least nine subclades (1). DNA photolyases repair cytotoxic and mutagenic DNA lesions that are formed during exposure of DNA to UV-B. These DNA lesions are cyclobutane pyrimidine dimers (CPDs) or pyrimidine-pyrimidone (6-4) photoproducts. According to their substrate specificity, DNA photolyases are designated as CPD photolyases or (6-4) photolyases (2). The repair of both types of DNA lesions by photolyase requires the catalytic fully reduced and anionic flavin cofactor FADH⁻ that, when photoexcited, injects an electron directly into the DNA lesion (1) as shown in Fig. 1A (electron transfer pathway 1). During extraction from the cell and purification under aerobic conditions the flavin cofactor is usually oxidized to the semireduced and eventually to the fully oxidized form. Reduction of these flavin species to FADH⁻ in vitro can be achieved by illumination of the enzyme in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol. This process is named photoactivation (1). Photoactivation in vitro requires photoexcitation of the flavin and a triad of redox-active residues in the protein moiety that is highly conserved in DNA photolyases (3, 4) as shown in Fig. 1A (electron transfer pathway 2). These residues are generally tryptophans that allow transport of an electron from the protein surface to the U-shaped flavin cofactor, which is buried within the C-terminal α-helical domain (5–9). Whether the same mechanism is used by photolyase to photoreduce FAD in vivo is a matter of debate (10). Photoreduction of the flavin cofactor was also observed in cryptochrome blue/UV-A photoreceptors. However, instead of fully reduced flavin, semireduced flavin species (either anionic flavin semiquinone radical or neutral semiquinone radical) accumulate. This form of the photoreceptor is considered as the signaling state (11–14).Open in a separate windowFIGURE 1.Electron transfer pathways in cry3 and structures of folates. A, indicated are the distances of the tryptophans in the tryptophan triad (Trp-356, -409, -432) of Trp-432 to FADH⁻ and of FADH⁻ to the 5,10-methenylTHF (MTHF) cofactor in cry3. Shown are also the two established routes of electrons from FADH⁻ to the DNA lesion (Route 1) and within the tryptophan triad to FAD (Route 2). The third electron transfer pathway from FADH⁻ to 5,10-methenylTHF (Route 3) is the subject of this study. B, chemical structures of folates and their molecular masses. Folypolyglutamate molecules have a pteridin and a p-aminobenzoate moiety linked with a glutamate chain with a variable number of glutamic acids. The various THF species differ in their oxidation state of the C1 unit that is attached at the N-5 or N-10 position or form a bridge between both.A recently discovered subclade of the DNA photolyase/cryptochrome family are DASH cryptochromes, which have members in plants, bacteria, and aquatic animals (6, 15–17). Because DASH cryptochromes were found to lack repair activity for CPDs in double-stranded DNA, they were considered as cryptochrome-type photoreceptors (6, 16). However, it was recently shown that DASH cryptochromes repair CPDs in single-stranded DNA (18) and loop structures of double-stranded DNA (19) and, thus, belong to the CPD photolyase group. In contrast to conventional CPD photolyases, DASH cryptochromes are unable to flip the CPD lesion out of the DNA duplex (7).Besides the flavin cofactor that is essential for enzymatic activity, DNA photolyases and most likely all cryptochromes contain a second chromophore (1). Like the catalytic flavin, the second chromophore is non-covalently attached to the protein moiety. The majority of DNA photolyases and, as far as studied, the cryptochromes including the DASH-type like cry3 from Arabidopsis thaliana contain polyglutamated 5,10-methenyltetrahydrofolate (5,10-methenylTHF) as the second chromophore (1, 12, 17, 20, 21) (see Fig. 1B for folate structures). Several organisms like the cyanobacterium Anacystis nidulans (Synechococcus elongatus) produce deazariboflavins (7,8-didemethyl-8-hydroxy-5-deazariboflavin) and utilize them as second cofactor (22). In photolyases of thermophilic bacteria and Archaea of the genus Sulfolobus, FMN and FAD, respectively, were found as second cofactors (23, 24). The sole function of the second cofactors demonstrated at present is transfer of excitation energy to the catalytic flavin cofactor via a Förster-type mechanism. The crystal structures of DNA photolyases and DASH cryptochromes revealed that the second chromophores are located in a cleft between the N-terminal α/β domain and the C-terminal α domain (7–9). The centroid distances between the catalytic FAD and the second chomophore are in the range of 15–18 Å. The close distances and the angles between the transition dipole moments of the two cofactors are favorable for efficient energy transfer. Indeed, energy transfer efficiencies are about 70% for Escherichia coli photolyase (25), close to 100% for A. nidulans photolyase (26), and between 78% (dark-adapted) and 87% (light-adapted) for Arabidopsis cry3 (27). Although the second cofactors are not essential for catalysis (28, 29), they increase the efficiency of repair and possibly of photoactivation by having higher extinction coefficients than FADH⁻ in the near UV and blue region (30). The spectral overlap between 5,10-methenylTHF emission and the absorption of the different flavin redox states is on the order FADH^o > FAD_ox > FADH⁻ (31).Illumination in vitro of photolyase that contains fully oxidized or semireduced flavin results in light-induced absorbance changes. The decrease in absorption in the 450–470-nm region reflects a decrease in the amount of fully oxidized FAD concomitant with transient increase in absorption above 500 nm, which indicates the formation of a neutral semiquinone radical. Excitation of the 5,10-methenylTHF antenna chromophore at its absorption peak at 380 nm causes a likewise photoreduction of the catalytic FAD (1, 27, 28, 30, 31). However, irreversible bleaching of the 380-nm peak is observed under high irradiance UV-A or camera flash illumination (28, 30). This irreversible bleaching goes along with release of the folate cofactor from the protein moiety (30) and was named photodecomposition of 5,10-methenylTHF (28). However, the identity of the formed folate species remained unknown (30). In our previous spectroscopic characterization of Arabidopsis cry3, a similar bleaching of the 380-nm peak was observed (27).Here we show that a third electron transfer pathway exists in photolyase and DASH cryptochome, where the 5,10-methenylTHF cofactor is photoreduced to 5,10-methyleneTHF. Thus, bleaching at 380 nm does not simply reflect destruction but is a specific chemical conversion of the second chromophore.     

Designing a biocontainment unit to care for patients with serious communicable diseases: a consensus statement

In spite of great advances in medicine, serious communicable diseases are a significant threat. Hospitals must be prepared to deal with patients who are infected with pathogens introduced by a bioterrorist act (e.g., smallpox), by a global emerging infectious disease (e.g., avian influenza, viral hemorrhagic fevers), or by a laboratory accident. One approach to hazardous infectious diseases in the hospital setting is a biocontainment patient care unit (BPCU). This article represents the consensus recommendations from a conference of civilian and military professionals involved in the various aspects of BPCUs. The role of these units in overall U.S. preparedness efforts is discussed. Technical issues, including medical care issues (e.g., diagnostic services, unit access); infection control issues (e.g., disinfection, personal protective equipment); facility design, structure, and construction features; and psychosocial and ethical issues, are summarized and addressed in detail in an appendix. The consensus recommendations are presented to standardize the planning, design, construction, and operation of BPCUs as one element of the U.S. preparedness effort.     

The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-ribosomal lipopeptide

Many secondary metabolic peptides from bacteria and fungi are produced by non-ribosomal peptide synthetases (NRPS) where the final step of biosynthesis is often catalysed by designated thioesterase domains. Here, we report the 1.8A crystal structure of the fengycin thioesterase (FenTE) from Bacillus subtilis F29-3, which catalyses the regio- and stereoselective release and macrocyclization of the antibiotic fengycin from the NRPS template. A structure of the PMSF-inactivated FenTE domain suggests the location of the oxyanion hole and the binding site of the C-terminal residue l-Ile11 of the lipopeptide. Using a combination of docking, molecular dynamics simulations and in vitro activity assays, a model of the FenTE-fengycin complex was derived in which peptide cyclization requires strategic interactions with residues lining the active site canyon.     

Structural and functional characterization of a synthetically modified OmpG

Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable β-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG–dansyl hybrid at 2.4 Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.     

Long-Term Positive and Negative Psychological Late Effects for Parents of Childhood Cancer Survivors: A Systematic Review

Increasing survival rates in childhood cancer have yielded a growing population of parents of childhood cancer survivors (CCSs). This systematic review compiles the literature on positive and negative long-term psychological late effects for parents of CCSs, reported at least five years after the child''s diagnosis and/or two years after the end of the child''s treatment. Systematic searches were made in the databases CINAHL, EMBASE, PsycINFO, and PubMed. Fifteen studies, published between 1988 and 2010, from 12 projects were included. Thirteen studies used quantitative methodology, one quantitative and qualitative methodology, and one qualitative methodology. A total of 1045 parents participated in the reviewed studies. Mean scores were within normal ranges for general psychological distress, coping, and family functioning. However, a substantial subgroup reported a clinical level of general psychological distress, and 21–44% reported a severe level of posttraumatic stress symptoms. Worry, disease-related thoughts and feelings, marital strains, as well as posttraumatic growth was reported. Several factors were associated with the long-term late effects, such as parents'' maladaptive coping during earlier stages of the childs disease trajectory and children''s current poor adjustment. Quality assessments of reviewed studies and clinical implications of findings are discussed and recommendations for future research are presented.     

Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC 6803

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.     

Bone cells from patients with quiescent Crohn's disease show a reduced growth potential and an impeded maturation

Patients with Crohn's disease (CD) are at increased risk of developing osteoporosis. The mechanism underlying bone loss in CD patients is only partly understood. Inflammation is thought to contribute by causing a disturbed bone remodeling. In this study, we aimed to compare functional characteristics of osteoblasts from CD patients and controls, as osteoblasts are one of the effector cells in bone remodeling. The study included 18 patients with quiescent CD and 18 healthy controls. Bone cells obtained from iliac crest biopsies were cultured in the absence and presence of the inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-10, and TGF-β. At various time points, cell proliferation and differentiation were analyzed. Bone cells from CD patients showed a prolonged culture period to reach confluence and a decreased cell number at confluence. CD patient-derived bone cell cultures produced higher alkaline phosphatase levels, whereas osteocalcin levels were considerably reduced compared to control cultures. At the proliferation level, the responsiveness to inflammatory cytokines was similar in bone cells from CD patients and controls. At the differentiation level, CD cultures showed an increased responsiveness to IL-6 and a decreased responsiveness to TGF-β. Responsiveness to the other cytokines tested was unaffected. In summary, we show a reduced growth potential and impeded maturation of bone cells from quiescent CD patients in vitro. These disease-related alterations combined with an unchanged sensitivity of CD patient-derived bone cells to inflammatory cytokines, provide a new insight in the understanding of CD-associated bone loss.     

CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors

Cryptochromes and photolyases are structurally related but have different biological functions in signalling and DNA repair. Proteobacteria and cyanobacteria harbour a new class of cryptochromes, called CryPro. We have solved the 2.7 Å structure of one of its members, cryptochrome B from Rhodobacter sphaeroides, which is a regulator of photosynthesis gene expression. The structure reveals that, in addition to the photolyase‐like fold, CryB contains two cofactors only conserved in the CryPro subfamily: 6,7‐dimethyl‐8‐ribityl‐lumazine in the antenna‐binding domain and a [4Fe‐4S] cluster within the catalytic domain. The latter closely resembles the iron–sulphur cluster harbouring the large primase subunit PriL, indicating that PriL is evolutionarily related to the CryPro class of cryptochromes.