Crystal structure of AdoMet radical enzyme 7‐carboxy‐7‐deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe–4S] cluster engender flavodoxin specificity

7‐Carboxy‐7‐deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7‐deazaguanine natural products. QueE is a member of the S‐adenosyl‐L‐methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX₃CX?C motif, to bind a [4Fe‐4S] cluster, and a partial (β/α)₆ TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster‐binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re‐haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7‐carboxy‐7‐deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.     

In vitro antileukemic activity of novel adenosine derivatives bearing boron cluster modification

A series of adenosine derivatives bearing a boron cluster were synthesized and evaluated for their cytotoxicity against primary peripheral mononuclear cells from the blood of 17 patients with leukemias (16 CLL and 1 very rare PLL), as well as from 5 healthy donors used as a control. Among the tested agents, two, i.e., compounds 1 and 2, displayed high in vitro cytotoxicity and proapoptotic potential on leukemic cells, with only scarce activity being seen against control cells. Biological tests related to apoptosis revealed the activation of the main execution apoptotic enzyme, procaspase-3, in CLL and PLL cells exposed to compounds 1 and 2. Moreover, the above compounds indicated high activity in the proteolysis of the apoptotic markers PARP-1 and lamin B1, fragmentation of DNA, and the induction of some changes in the expression of the Mcl-1, protein apoptosis regulator in comparison with control cells.     

The impact on high‐grade serous ovarian cancer of obesity and lipid metabolism‐related gene expression patterns: the underestimated driving force affecting prognosis

To investigate whether specific obesity/metabolism‐related gene expression patterns affect the survival of patients with ovarian cancer. Clinical and genomic data of 590 samples from the high‐grade ovarian serous carcinoma (HGOSC) study of The Cancer Genome Atlas (TCGA) and 91 samples from the Australian Ovarian Cancer Study were downloaded from the International Cancer Genome Consortium (ICGC) portal. Clustering of mRNA microarray and reverse‐phase protein array (RPPA) data was performed with 83 consensus driver genes and 144 obesity and lipid metabolism‐related genes. Association between different clusters and survival was analyzed with the Kaplan–Meier method and a Cox regression. Mutually exclusive, co‐occurrence and network analyses were also carried out. Using RNA and RPPA data, it was possible to identify two subsets of HGOSCs with similar clinical characteristics and cancer driver mutation profiles (e.g. TP53), but with different outcome. These differences depend more on up‐regulation of specific obesity and lipid metabolism‐related genes than on the number of gene mutations or copy number alterations. It was also found that CD36 and TGF‐ß are highly up‐regulated at the protein levels in the cluster with the poorer outcome. In contrast, BSCL2 is highly up‐regulated in the cluster with better progression‐free and overall survival. Different obesity/metabolism‐related gene expression patterns constitute a risk factor for prognosis independent of the therapy results in the Cox regression. Prognoses were conditioned by the differential expression of obesity and lipid metabolism‐related genes in HGOSCs with similar cancer driver mutation profiles, independent of the initial therapeutic response.     

Context-Specific Mechanisms of Cell Polarity Regulation

Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses. The polarity protein complexes regulating these interactions are highly evolutionarily conserved between vertebrates and invertebrates. Interestingly, these polarity complexes interact with each other and key signaling pathways in a cell-polarity context-dependent manner. However, the exact mechanisms by which these interactions take place are poorly understood. In this review, we will focus on the roles of the key polarity complexes SCRIB, PAR and Crumbs in regulating different forms of cell polarity, including epithelial cell polarity, cell migration, asymmetric cell division and the T-cell immunological synapse assembly and signaling.     

Generation of semiquinone-[2Fe-2S]+ spin-coupled center at the Qo site of cytochrome bc1 in redox-poised,illuminated photosynthetic membranes from Rhodobacter capsulatus

One of the less understood parts of the catalytic cycle of cytochrome bc₁/b₆f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Q_o site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc₁ or b₆f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Q_o site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.     

Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis

The dynamics of enzyme catalysis range from the slow time scale (～ms) for substrate binding and conformational changes to the fast time (～ps) scale for reorganization of substrates in the chemical step. The contribution of global dynamics to catalysis by alcohol dehydrogenase was tested by substituting five different, conserved amino acid residues that are distal from the active site and located in the hinge region for the conformational change or in hydrophobic clusters. X‐ray crystallography shows that the structures for the G173A, V197I, I220 (V, L, or F), V222I, and F322L enzymes complexed with NAD⁺ and an analogue of benzyl alcohol are almost identical, except for small perturbations at the sites of substitution. The enzymes have very similar kinetic constants for the oxidation of benzyl alcohol and reduction of benzaldehyde as compared to the wild‐type enzyme, and the rates of conformational changes are not altered. Less conservative substitutions of these amino acid residues, such as G173(V, E, K, or R), V197(G, S, or T), I220(G, S, T, or N), and V222(G, S, or T) produced unstable or poorly expressed proteins, indicating that the residues are critical for global stability. The enzyme scaffold accommodates conservative substitutions of distal residues, and there is no evidence that fast, global dynamics significantly affect the rate constants for hydride transfers. In contrast, other studies show that proximal residues significantly participate in catalysis.     

Propagation and tuberization of potato bud clusters from bioreactor culture

Summary Single node stem segments fromin vitro potato shoots cultured in liquid medium in the presence of ancymidol (23.4 μM) developed into bud clusters in either shaken flasks or bioreactor cultures. Buds on the clusters developed tubers after subculture to a tuber induction medium with 23.2 μM kinetin, 19.5 μM ancymidol, and 6-8% sucrose. The number of tubers per cluster and their size were higher in agar induction medium on top of which a second layer of liquid medium was added, than in liquid shake or bioreactor cultures. The highest increase in tuber size (i.e., 720 mg fresh weight after 7 weeks), was obtained in agar cultures flushed twice with liquid tuber induction medium. The potential of bioreactor cultures for potato bud proliferation and enhanced tuber development in double layer agar-liquid cultures is discussed.     

Freeze-protection of overwintering monarch butterflies in Mexico: critical role of the forest as a blanket and an umbrella

Abstract. 1. At their high-altitude overwintering sites in Mexico, monarch butterflies frequently are subjected to sub-zero°C temperatures during December-March. Although monarchs have moderate supercooling ability, two ecological factors strongly influence their capacity to resist freezing: wetting and exposure to the clear night sky. 2. As shown in Fig. 2, 50% of a population of butterflies with water on their body surfaces freeze at warmer sub-zero temperatures (-4.2°C) compared to butterflies with no water on their bodies (-7.7°C). 100% mortality occurs, respectively, at ?7.7°C and ?15°C. 3. Comparative measurements of rainfall within a large overwintering colony in Mexico indicated that the intact canopy acts as an umbrella that reduces butterfly wetting during winter storms. 4. Variable experimental exposure of butterflies to the clear night sky indicated that openings in the forest canopy increases radiational cooling and causes monarch body temperatures to drop as much as 4°C below ambient air temperature. Monarchs under dense cover had body temperatures approximately the same as the ambient air temperature, but more exposed individuals had body temperatures below ambient in direct proportion to the degree of exposure. Consequently, forest thinning increases the probability that the butterflies will freeze to death. 5. Whereas both wetting and exposure are increased by disturbance of the forest canopy, the interaction of these two factors exacerbates freezing mortality during winter storms: 50% of dry and unexposed butterflies froze at ?8°C, whereas wetted and fully exposed butterflies froze at only ?0.5°C. 6. Butterflies inside and on the bottom of the fir bough clusters are better protected from wetting than those on the outside. This supports the hypothesis that the structure of the butterfly clusters has evolved through individual selection to avoid wetting. 7. The data strongly reinforce previous evidence that forest thinning should be totally prevented within and adjacent to the overwintering sites in order to minimize both wetting and exposure of the butterflies that synergistically increase winter mortality at the overwintering sites in Mexico.