The Dimerization Domain in DapE Enzymes Is Required for Catalysis

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,Ldiaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate

Effects of mechanical injury and insect feeding on volatiles emitted by wheat plants

We report on changes in the volatiles emitted by wheat plants of the cultivar ‘Tonacja’ after the plants were scraped, pierced or damaged by adults of Oulema melanopus (Linnaeus). The blend of volatiles was dominated by typical green leaf volatiles and in addition contained linalool and B-caryophyllene, but the latter two were released in large amounts only after more than one day of insect feeding. Overall, the plants that had been damaged by the insect released more odors than the plants that were subjected to mechanical damage. Moreover, control plants released significantly lower amounts of volatiles. Scraping of leaves induced higher volatile emission than puncturing the leaves. A Y-tube bioassay was used to evaluate upwind orientation ofadult cereal leafbeetles. Greater number of female 0. melanopus was attracted to (Z)-3-hexenal and (Z)-3 -hexenyl acetate at low concentrations. Higher concentration of these compounds and linalool significantly repelled both females and males

Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn–Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes

Alteration of parafollicular (C) cells activity in the experimental model of hypothyroidism in rats.

Our previous study has shown the alteration of C cells activity in rats with experimental model of hyperthyroidism. The aim of the present study was the evaluation of parafollicular cells activity in rats with hypothyroidism evoked by propylthiouracil (PTU) given in drinking water over 21 days. Histological, ultrastructural and immunocytochemical studies using specific antibodies against calcitonin and CGRP were performed on thyroid glands taken from experimental and control groups of rats. Moreover, in all animals the calcitonin plasma levels were evaluated by radioimmunoassay. After chronic administration of PTU, thyroid image showed predominant microfollicular hyperplasia and attenuated density of parafollicular cells. The intensity of immunocytochemical reactions for CT and CGRP were weaker in the majority of C cells in comparison to the control rats, in which strong immunocytochemical reaction was observed. Examination in the electron microscope reveals the features of hypoactivity both in follicular and parafollicular cells, in which the quantity and electron density of secretory granules were smaller in comparison to the control group. These microscopic changes were accompanied by a significant decrease of calcitonin plasma concentration. Alteration of C cells activity in the experimental model of hypothyroidism, accompanied by microfollicular hypertrophy, may point to the mutual cooperation between parafollicular and follicular cells

A Shift from 2D Design Paradigm of the 19 th Century to 3D/CityGML, BIM, 3D Printing and Some of Smarter Cities in Poland

A road from paper-based-administration of the 80-ties to Smart Cities of today is being showed in this paper. Shift from paper do digital environment started with regaining of Polish independence in 1989, 26 years ago. The first e-mail from Poland was sent in 1990 year, 19 years after the first e-mail on the world of Ray Tomlinson (1971). Transfter of legal responsibilities, legal power, competences and finance from the top to local levels resulted in revolution in IT sector, which was the first commercial sector running in apost comunist country, in the 80-ties and the beginning of 90ties.Pressure for changes was visible exspecially in the biggest cities, and were initially connected with process of “mucicipalization” – i.e. – transfering of ownership of land from the state level to the level of municipalities. Signum Tempori of this time, was a process of transfer of state owned land to the city property of the City of Gdansk, where more than 30000 real estates of of the market value of 750 million US dollars were transferred and became municipal between 1992 and 1994 years. More and more LIS (Land Information Systems) and GIS (Geographic Information Systems) were implemented, without interoperability rules and standards. Lack of ability to adapt centrain common standards between State Surveying and the biggest cities resulted in appearance of more than 20 graphical applications and more then 20 textual databases applications which required later substantial efforts and costs to overcome information chaos. 10 biggest metropolitan Polish Cities spend more then 3 times than the General Office of Geodesy and Cadasrte of Poland, between 1991 and 1994. Gradual implementaion of INSPIRE Directive and the Law of National Infrastrructure of Spatial Information created unprecedented shitf from paper maps and paper records to almost all digital Poland. Expenditures of c.a. 650 million PLN were assigned to creation of digital representaion of all 34 data layers of INSPIRE Directive for the impelmentaion period of 2010 to 2019. Nevertheless, this amount has been almost doubled in the first 3 years, taking into acccount expenditures of only regional and local GIS/SDI Projects. Polish spatial and economic conditions created spatio-economic background, within which more than 65 % of GDP of Poland is located within 12 metropolitan areas, and at the same time around 67 % of Polish GDP is being generated by more than 4 million of micro or small businesses (often small “family” businesses.).Polish Spatial Planning Law of 2003 has weakened spatial planning regulation, allowing for certain “exception from the rule”, which became a new rule in itself. Basically, this “door” in the law to obtain building permint outside the borders of local development plan – resulted in issuing of more than 700 000 building permits – all located ouside territories of local spatial development plans between 2003 and 2015. Therefore we observe freely flowing process of urban sprawl on one hand and increased land consumption, expecially in the peri-urban zones of all metropolitan cities, and on the other hand, from the economic point of view – Poland has experienced unprecedented GDP growth in recent 10 or 12 years. Nevertheless several really interesting projects have been kicked-off by metropolitan cities, regions and General Survey of Poland (GUGIK). One of the most interesting projects – ISOK (Informatic System of State Protection against Extraordinary Threads) was impelmented between 2011 and 2015, at the cost of c.a. 300 million PLN, resulting in creation of laser scanning data for 92 % of territory of Poland. Continuation of this project was secured in the autumn of 2015 year, devoting budget of 189 million PLN for the project called CAPAP (acronyme from “ Centre of Spatial Analysis of Public Administration), which aim is to provide 3D model of all buildings in Poland, in compliance with CityGML LOD 2 (second Level of Detail), withing the time frame 2016 – 2018. So, all territory of Poland will become 3D in 3 years time in accordance with CityGLM LoD2 and some studies and pilot projects going in this direction are being described in this paper. Some recent exercises with 3D printing of new urban projects are being reported at the end of article

Interleukin-6 is not essential for bone turnover in hypothyroid mice.

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by an imbalance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) and osteocalcin in IL-6-deficient mice to assess the role of IL-6 in bone metabolism in hypothyroidism in mice. C57BL/6J (wild-type; WT) and C57BL/6J(IL6-/-Kopf) (IL-6 knock-out; IL6KO) mice randomly divided into 4 groups with 10 in each one: 1/ WT mice in hypothyroidism (WT-ht), 2/ WT controls, 3/ IL6KO mice with hypothyroidism (IL6KO-ht) and 4/ IL6KO controls. Experimental model of hypothyroidism was induced by intraperitoneal injection of propylthiouracyl. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentrations of TRACP 5b (median and interquartile ranges) were significantly decreased in both groups of mice with hypothyroidism: WT (3.2 (2.5-4.7) U/l) and IL6KO (2.6 (1.8-3.5) U/l) as compared to the respective controls. Similarly, serum osteocalcin levels were significantly reduced in both groups of mice in experimental hypothyroidism: WT (25.8 (23.0-28.2) ng/ml) and IL6KO (21.5(19.0-24.6) ng/ml) in comparison to the respective controls. There were no significant differences in bone turnover markers between IL6KO and WT mice both in hypothyroid and control animals. The results of the present study suggest that IL-6 does not play an important role in bone turnover in both euthyroid and hypothyroid mice

Crystal Structure of a Nonsymbiotic Plant Hemoglobin

Background: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O2 conditions. Results: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 Å resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b5. The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. Conclusions: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O2 reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O2 affinity is ultra-high (KD ≈ 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified

Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO₂ as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from <i>Alcanivorax borkumensis</i> and RPA1511 from <i>Rhodopseudomonas palustris</i>. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and <i>p</i>-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling