Structural and biophysical studies of new L-asparaginase variants : lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amido­hydrolase

This work reports the results of random mutagenesis of the Escherichia coli class 2 l-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the l-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for l-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes

SOLARIS National Synchrotron Radiation Centre in Krakow, Poland

The SOLARIS synchrotron located in Krakow, Poland, is a third-generation light source operating at medium electron energy. The first synchrotron light was observed in 2015, and the consequent development of infrastructure lead to the first users’ experiments at soft X-ray energies in 2018. Presently, SOLARIS expands its operation towards hard X-rays with continuous developments of the beamlines and concurrent infrastructure. In the following, we will summarize the SOLARIS synchrotron design, and describe the beamlines and research infrastructure together with the main performance parameters, upgrade, and development plans

Structural and functional studies of plant-type asparaginases and their novel variants.

Ostra białaczka limfoblastyczna (ALL) jest najczęściej występującym nowotworem złośliwym u dzieci. Jednym z chemioterapeutyków wykorzystywanych w terapii ALL jest L-asparaginaza. Obecnie w terapii wykorzystywane są enzymy pochodzące z bakterii E. coli (EcAII) oraz D. dadantii. Stosowanie tych preparatów obarczone jest jednak szeregiem efektów ubocznych. Inną grupą L-asparaginaz, zidentyfikowanych u E. coli (EcAIII) są enzymy typu roślinnego. Enzymy z tej grupy należą do Ntn-hydrolaz. Jednak ich powinowactwo do L-asparaginy jest zbyt niskie, aby wykorzystać je w leczeniu ALL. Jednym ze sposobów poszukiwania enzymów o pożądanych właściwościach jest mutageneza przypadkowa. Wykorzystując ją można przygotować bibliotekę wariantów, wśród których można poszukiwać białek o najlepszych właściwościach. Takie działanie może w przyszłości pomóc stworzyć L-asparaginazę cechującą się zwiększoną skuteczność kliniczną, przy jednoczesnym zminimalizowaniu efektów ubocznych. W pracy opisano produkcję i charakterystykę rekombinowanych wariantów EcAIII. Badane enzymy posiadały mutację w rejonach miejsca aktywnego EcAIII. Wyniki pokazały, że mutacje wpływają na zdolności do krystalizacji, strukturę drugorzędową, stabilność termiczną i aktywność enzymatyczną białek.Acute lymphoblastic leukemia (ALL) is the most common acute childhood cancer. One of the chemotherapeutic agents used to treat ALL is L-asparaginase. Currently, only enzymes from E. coli (EcAII) and E. dadantii are used in the therapy. However, they are responsible for serious side effects. Another group of L-asparaginases consists of plant-type enzymes identified e.g. in E. coli (EcAIII). Plant-type enzymes belong to the Ntn-hydrolases, however, their affinity for L-asparagine is too low to make them a successful therapeutics used in ALL treatment. A random mutagenesis is a common approach used to designing enzymes with specific properties. It allows to create a library of mutants and select clones with the best properties. This approach also can be used to create a new type of L-asparaginase, with good clinical efficiency and minimized side effects. This study describes the production and characterization of recombinant EcAIII and its new variants. The examined enzymes had mutations in the region of the active site. The results showed that mutations affect protein crystallization process, secondary structure, thermal stability and enzymatic activity

Photoactivity of nanomaterials on DNA.

Szybki rozwój i coraz większe zainteresowanie nanocząstkami i ich zastosowaniem w przemyśle, technologii oraz medycynie wymaga przebadania ich wpływu na środowisko, procesy biologiczne i organizm człowieka. Ich nanometryczne rozmiary i unikalne właściwości, takie jak duży stosunek powierzchni do masy, mogą skutkować między innymi znaczną reaktywnością. Jednak mimo sporego zainteresowania tymi materiałami niewiele wiadomo na temat ich potencjalnej toksyczności. Część nanomateriałów może mieć charakter półprzewodnikowy, co może skutkować powstaniem toksycznych efektów po wystawieniu na promieniowanie świetlne. Istnieje wiele rodzajów nanocząstek, a ich duża powierzchnia właściwa umożliwia stworzenie różnych kombinacji i modyfikacji powierzchniowych, co warunkuje ich zróżnicowane działanie. W pracy przedstawiony został wpływ wybranych nanocząstek, nanocząstek modyfikowanych powierzchniowo, światła widzialnego oraz światła UVA na modelowy materiał genetyczny.Increasing more interest in nanoparticles, their rapid development year to year and application in industry, technology and medicine induced research on their impact on the human body and the environment. Their nanoscale sizes and unique properties (for example high specific surface to mass ratio) can result high activity. However, despite considerable interest in these materials little is known about their toxicity. There are many types of nanoparticles, and their large specific surface area allows the creation of various combinations and surface modifications, which determines their varied performance. Some of these materials may be semiconductor, which may result in toxic effects when exposed to light radiation. This work presents the impact of selected nanoparticles, surface-modified nanoparticles, visible light and UVA light on model genetic material

Structural and biophysical studies of new L -asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes