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.
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.
Since its introduction in the early 1970s, high pressure crystallography (HPX) has shown great potential for the investigation of different types of matter. Using diamond anvil cells, HPX is an emerging technique that has been rapidly implemented, making it available to biologists, and there is immense potential for utilizing this technique in biological systems in the future. At the molecular level, high-pressure crystallographic investigation provides information on structural characteristics that not only determine the native conformation of a protein but also the conformations with higher free-energy, thus revealing function-related structural changes and properties that can be modified as a result of pressurization. The increase in the number of crystal structures of different macromolecules determined under high pressure over the last five decades can be ascribed mainly to two factors: the emergence of high-pressure cells with very large, open angles, and the advent of third generation synchrotron sources. The use of high pressure crystallography as a research tool has been shown to contribute to the advancements in the basic fields of biochemistry (protein misfolding and aggregation), biophysics (protein stability), and biotechnology (food processing). Presently, with a growing interest in biomedicine and nanotechnology, this nonstandard method appears to be a valid instrument for probing more challenging and complex systems. In this review, we present the method, highlight a selection of recent applications, and describe challenges for high pressure macromolecular crystallography (HPMX).
L-asparaginases are used in the treatment of Acute Lymphoblastic Leukemia (ALL). However, the currently utilized bacterial-type enzymes cause several severe side effects. Therefore, there is an urgent need to develop a new generation of therapeutic Lasparaginases. Promising candidates can be found among plant-type L-asparaginases, which function as Ntn-hydrolases. Ntn-hydrolases are produced as inactive precursors and develop enzymatic activity in an autoproteolytic maturation process. Unfortunetely, therapeutic use of wild-type (WT) plant-type enzymes is not possible due to their low substrate affinity (mM). Random mutagenesis is a protein engineering tool that can be used to enhance enzyme efficiency and study structure-function relationship. In this project, locally performed random mutagenesis was used to generate a library of mutants of plant-type L-asparaginase from E.coli (EcAIII). Several new variants of EcAIII were selected from the library and subjected to detailed structural (X-ray crystallography) and biophysical studies (nanoDSF, CD, activity/autoproteolytic tests).
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