Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca²⁺ and/or Mg²⁺, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca²⁺/Mg²⁺ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca²⁺ and 2.5 mM Mg²⁺ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca²⁺/Mg²⁺. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca²⁺/Mg²⁺. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.     

Cadmium decontamination and reversal potential of teratological forms of the diatom Planothidium frequentissimum (Bacillariophyceae) after experimental contamination

While the induction of teratology by cadmium (Cd) on diatoms is already known, reversal kinetics are not well documented. This study aims to understand the viability of diatoms exhibiting teratological frustules and their reproduction capacities within a Cd‐impacted population to predict their return to normal diatom forms. We worked on a frequently encountered species in French hydrosystems: Planothidium frequentissimum (Lange‐Bertalot) Round & L. Bukhtiyarova. First, a 21‐d contamination phase highlighted increasing inductionof different teratological types in response to two levels of Cd contamination: 20 and 100 μg · L^?1. The deformity counting indicated that Cd firstly generated striae and mixed teratologies, then affected the central area and the valves. Second, a 28‐d decontamination phase demonstrated the Cd depuration capacity of Planothidium frequentissimum. Cd half‐lives appeared relatively low, ~6 d for the 100 μg · L^?1 condition. Moreover, the decontamination phase showed a decrease in teratology abundances, but a still incomplete recovery after 28 d. Deformations of the striae appeared to be the most sustainable phenotype since they were still significantly higher than in reference cultures at the end of the decontamination phase for both Cd cultures.     

Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Diversity and evolution of the highly polymorphic tandem repeat LEI0258 in the chicken MHC-B region

The chicken major histocompatibility complex (MHC) is located on the microchromosome 16 and is described as the most variable region in the genome. The genes of the MHC play a central role in the immune system. Particularly, genes encoding proteins involved in the antigen presentation to T cells. Therefore, describing the genetic polymorphism of this region is crucial in understanding host–pathogen interactions. The tandem repeat LEI0258 is located within the core area of the B region of the chicken MHC (MHC-B region) and its genotypes correlate with serology. This marker was used to provide a picture of the worldwide diversity of the chicken MHC-B region and to categorize chicken MHC haplotypes. More than 1,600 animals from 80 different populations or lines of chickens from Africa, Asia, and Europe, including wild fowl species, were genotyped at the LEI0258 locus. Fifty novel alleles were described after sequencing. The resulting 79 alleles were classified into 12 clusters, based on the SNPs and indels found within the sequences flanking the repeats. Furthermore, hypotheses were formulated on the evolutionary dynamics of the region. This study constitutes the largest variability report for the chicken MHC and establishes a framework for future diversity or association studies.     

The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis

1. Download : Download high-res image (196KB)
2. Download : Download full-size image

Highlights? Yeast Hsp70 SSB binds cotranslationally to a defined set of nascent polypeptides ? SSB shows specificity for substrates highly challenged in cotranslational folding ? The heterodimeric cochaperone RAC modulates cotranslational SSB specificity ? Loss of SSB function leads to widespread misfolding and aggregation     

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.     

GH–IGF system regulation of attenuated muscle growth and lipolysis in Atlantic salmon reared at elevated sea temperatures

Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 °C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 °C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at ≤17 °C, and dropped in fish reared at 19 °C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 °C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 °C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 °C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 °C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 °C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.