Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

Virus membrane-fusion proteins: more than one way to make a hairpin

Structure-function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. In both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. This review will focus in particular on the properties of the more recently described class II proteins.     

Evaluation of <Superscript>15</Superscript>N-detected H–N correlation experiments on increasingly large RNAs

Recently, ¹⁵N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a ¹⁵N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three ¹⁵N-detected H–N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established ¹⁵N-detected BEST-TROSY experiment, which proves to be the most sensitive ¹⁵N-detected H–N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the ¹⁵N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the ¹H- than in the ¹⁵N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that ¹⁵N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.     

Recognition of anionic phospholipid membranes by an antihemostatic protein from a blood-feeding insect

The saliva of blood-feeding insects contains a variety of molecules having antihemostatic activity. Here, we describe nitrophorin 7 (NP7), a salivary protein that binds with high affinity to anionic phospholipid membranes. The protein is apparently targeted to the negatively charged surfaces of activated platelets and other cells, where it can serve as a vasodilator, antihistamine, platelet aggregation inhibitor, and anticoagulant. As with other members of the nitrophorin group, NP7 reversibly binds a molecule of NO and binds histamine with high affinity. The protein differs from other nitrophorins in that it binds to membranes containing phosphatidylserine. Sedimentation and surface plasmon resonance experiments, revealed two classes of phospholipid-binding sites having K(d) values of 4.8 and 755 nM. NP7 inhibits prothrombin activation by blocking phospholipid binding sites for the prothrombinase complex on the surfaces of vesicles and activated platelets. As a NO complex, NP7 inhibits collagen and ADP-induced platelet aggregation and induces disaggregation of ADP-stimulated platelets by an NO-mediated mechanism. Molecular modeling of NP7 revealed a putative, positively charged membrane interaction surface comprised mainly of a helix lying outside of the lipocalin beta-barrel structure.     

The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein–protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.     

Biomass change in an Atlantic tropical moist forest: the ENSO effect in permanent sample plots over a 22-year period

There are a number of controversies surrounding both biomass estimation and carbon balance in tropical forests. Here we use long-term (from 1978 through 2000) data from five 0.5-ha permanent sample plots (PSPs) within a large tract of relatively undisturbed Atlantic moist forest in southeastern Brazil to quantify the biomass increment (M_I), and change in total stand biomass (M_stand), from mortality, recruitment, and growth data for trees 10 cm diameter at breast height (DBH). Despite receiving an average of only 1,200 mm annual precipitation, total forests biomass (334.5±11.3 Mg ha^–1) was comparable to moist tropical forests with much greater precipitation. Over this relatively long-term study, forest biomass experienced rapid declines associated with El Niño events, followed by gradual biomass accumulation. Over short time intervals that overlook extreme events, these dynamics can be misinterpreted as net biomass accumulation. However for the 22 years of this study, there was a small reduction in forest biomass, averaging –1.2 Mg ha^–1 year^–1 (±3.1). Strong climatic disturbances can severely reduce forest biomass, and if the frequency and intensity of these events increases beyond historical averages, these changing disturbance regimes have the capacity to significantly reduce forest biomass, resulting in a net source of carbon to the atmosphere.     

Possible female philopatry of the smooth hammerhead shark Sphyrna zygaena revealed by genetic structure patterns

We assessed the spatial pattern of genetic structure of smooth hammerhead shark Sphyrna zygaena in 10 localities from the Northern Mexican Pacific. A total of 35 haplotypes were identified in 129 sequences of the mtDNA control region. The results showed slight but significant genetic structure among localities (Φ_ST = 0.044, P < 0.001). In addition, the localities with highest number of juveniles were genetically different (Φ_ST = 0.058, P < 0.024), which may be representative of nursery areas. The genetic differentiation pattern can be associated to female philopatry and preference for particular birthing sites. Finally, historical demography shows that S. zygaena populations present a recent demographic expansion that occurred during glacial events in the late Pleistocene to early Holocene.     

Fueling Plankton Production by a Meandering Frontal Jet: A Case Study for the Alboran Sea (Western Mediterranean)

A three dimensional biophysical model was employed to illustrate the biological impacts of a meandering frontal jet, in terms of efficiency and persistency of the autotrophic frontal production, in marginal and semi-enclosed seas. We used the Alboran Sea of the Western Mediterranean as a case study. Here, a frontal jet with a width of 15–20 km, characterized by the relatively low density Atlantic water mass, flows eastward within the upper 100 m as a marked meandering current around the western and the eastern anticyclonic gyres prior to its attachment to the North African shelf/slope topography of the Algerian basin. Its inherent nonlinearity leads to the development of a strong ageostrophic cross-frontal circulation that supplies nutrients into the nutrient-starved euphotic layer and stimulates phytoplankton growth along the jet. Biological production is larger in the western part of the basin and decreases eastwards with the gradual weakening of the jet. The higher production at the subsurface levels suggests that the Alboran Sea is likely more productive than predicted by the satellite chlorophyll data. The Mediterranean water mass away from the jet and the interiors of the western and eastern anticyclonic gyres remain unproductive.