Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications

Alpha-glucuronidases cleave the alpha-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the alpha-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the alpha-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp(364) and Glu(392), activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu(285) cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu(285) is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.     

A reciprocal single mutation affects the metal requirement of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthases from Aquifex pyrophilus and Escherichia coli

The enzyme 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys11 was replaced by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn26 to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (kcat/Km) was reduced by only approximately 12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn2+ or Cd2+ stimulated its activity to approximately 30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.     

Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application

Biotin is an essential cofactor for a variety of carboxylase and decarboxylase reactions and is involved in diverse metabolic pathways of all organisms. In the present study we tested the hypothesis that controlling biotin availability by the expression of Streptomyces avidinii streptavidin, would impede plant development. Transient expression of streptavidin fused to plant signal peptide, bacterial signal peptide or both, in tomato (Lycopersicon esculentum cv. VF36) plants resulted in various levels of tissue impairment, exhibited as lesion development on 1-week-old tomato seedlings. The least toxic construct was introduced to tomato (stable transformation) under the constitutive CaMV 35S promoter, and lesions appeared on stems, flower morphologies were modified and numbers and sizes of fruits were altered. Furthermore, tissue-specific expression of the streptavidin, by means of the beta-phaseolin or TobRB7 promoters, resulted in localised effects, i.e., impaired seed formation or seedless fruits, respectively, with no alteration in the morphology of the other plant organs. External application of biotin on streptavidin-expressing tomato plants prevented the degeneration symptoms and facilitated normal plant development. It can be concluded that expression of streptavidin in the plant cell can lead to local and temporal deficiencies in biotin availability, impairing developmental processes while biotin application restores plant growth cycle.     

Inulinase immobilization on polyethylene glycol/polypyrrole multiwall carbon nanotubes producing a catalyst with enhanced thermal and operational stability

This paper describes the development of a simple method for mixed non‐covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes (f‐MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N‐H functional groups from PPy and ‐COO^? from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345 μg protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV‐Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half‐life (t_1/2) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99 min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.     

Significant host- and environment-dependent differentiation among highly sporadic fungal endophyte communities in cereal crops-related wild grasses

Endophytic fungi compose a significant part of plant microbiomes. However, while a small number of fungal taxa have proven beneficial impact, the vast majority of fungal endophytes remain uncharacterized, and the drivers of fungal endophyte community (FEC) assembly are not well understood. Here, we analysed FECs in three cereal crops-related wild grasses – Avena sterilis, Hordeum spontaneum and Aegilops peregrina – that grow in mixed populations in natural habitats. Taxa in Ascomycota class Dothideomycetes, particularly the genera Alternaria and Cladosporium, were the most abundant and prevalent across all populations, but there was also high incidence of basidiomyceteous yeasts of the class Tremellomycetes. The fungal community was shaped to large extent by stochastic processes, as indicated by high level of variation even between individuals from local populations of the same plant species, and confirmed by the neutral community model and Raup-Crick index. Nevertheless, we still found strong determinism in FEC assembly with both incidence and abundance data sets. Substantial differences in community composition across host species and locations were revealed. Our research demonstrated that assembly of FECs is affected by stochastic as well as deterministic processes and suggests strong effects of environment heterogeneity and plant species on community composition. In addition, a small number of taxa had high incidence and abundance in all of the 15 populations. These taxa represent an important part of the core FEC and might be of general functional importance.     

The dialectic between modernization and orientalization: ethnicity and work relations in the 1950s Lakhish region project

This article focuses on agricultural planning and work relations in the Lakhish regional settlement project in 1950s Israel. During this period, Jewish immigrants from Arab countries – considered the other of allegedly Modern and Western Israeli society – were sent to settle the new frontier, in order to establish Jewish sovereignty over former Palestinian land. I discuss the ways in which agricultural planning and the organization of the work process created a relationship of dependency between the settlers and the settling institutions. I show, that the plans were shaped by Orientalist assumptions concerning the nature of the settlers, their social and family relationships and their cognitive abilities. I argue that it was these plans and the work relations they engendered, that doomed the settlers from the start, undermining their ability to form functioning cooperative communities. The settlers, however, were not passive objects of state policies but rather displayed patterns of resistance to state regulation.     

Morphology and function of the lumbar spine of the Kebara 2 Neandertal

The morphology of the lumbar spine is crucial for upright posture and bipedal walking in hominids. The excellent preservation of the lumbar spine of Kebara 2 provides us a rare opportunity to observe a complete spine and explore its functionally relevant morphology. The lumbar spine of Kebara 2 is analyzed and compared with the lumbar spines of modern humans and late Pleistocene hominids. Although no size differences between the vertebral bodies and pedicles of Kebara 2 and modern humans are found, significant differences in the size and orientation of the transverse processes (L₁‐L₄), and the laminae (L₅, S₁) are demonstrated. The similarity in the size of the vertebral bodies and pedicles of Kebara 2 and modern humans suggests similarity in axial load transmission along the lumbar spine. The laterally projected (L₂‐L₄) and the cranially oriented (L₁, L₃) transverse processes of Kebara 2 show an advantage for lateral flexion of the lumbar spine compared with modern humans. The characteristic morphology of the lumbar spine of Kebara 2 might be related to the wide span of its pelvic bones. Am J Phys Anthropol 142:549–557, 2010. © 2010 Wiley‐Liss, Inc.     

Perturbation analysis analyzed—mathematical modeling of intact and perturbed gene regulatory circuits for animal development

Gene regulatory networks for animal development are the underlying mechanisms controlling cell fate specification and differentiation. The architecture of gene regulatory circuits determines their information processing properties and their developmental function. It is a major task to derive realistic network models from exceedingly advanced high throughput experimental data. Here we use mathematical modeling to study the dynamics of gene regulatory circuits to advance the ability to infer regulatory connections and logic function from experimental data. This study is guided by experimental methodologies that are commonly used to study gene regulatory networks that control cell fate specification. We study the effect of a perturbation of an input on the level of its downstream genes and compare between the cis-regulatory execution of OR and AND logics. Circuits that initiate gene activation and circuits that lock on the expression of genes are analyzed. The model improves our ability to analyze experimental data and construct from it the network topology. The model also illuminates information processing properties of gene regulatory circuits for animal development.