Bhattarai, H. K. | Search bioRxiv

bioRxiv2020

In-silico development of a method for the selection of optimal enzymes using L-asparaginase II against Acute Lymphoblastic Leukemia as an example.

L-Asparaginase II (asnB), a periplasmic protein, commercially extracted from E. coli and Erwinia, is often used to treat Acute Lymphoblastic Leukemia. L-Asparaginase is an enzyme that converts L-asparagine to aspartic acid and ammonia. Cancer cells are dependent on asparagine from other sources for growth and when these cells are deprived of asparagine by the action of the enzyme the cancer cells selectively die. Questions remain as to whether asnB from E. coli and Erwinia is the best asparaginase as they have many side-effects. asnB with the lowest Michaelis constant (Km) (most potent), and with the lowest immunogenicity is considered the most optimal enzyme. In this paper asnB sequence of E. coli was used to search for homologous proteins in different bacterial and archaeal phyla and a maximum likelihood phylogenetic tree was constructed. The sequences that are most distant from E. coli and Erwinia were considered best candidates in terms of immunogenicity and were chosen for further processing. The structures of these proteins were built by homology modeling and asparagine was docked with these proteins to calculate the binding energy. asnBs from Streptomyces griseus, Streptomyces venezuelae and Streptomyces collinus were found to have the highest binding energy i.e. -5.3 kcal/mol, -5.2 kcal/mol, and -5.3 kcal/mol respectively (Higher than the E.coli and Erwinia asnBs) and were predicted to have the lowest Kms as we found that there is an inverse relationship between binding energy and Km. Besides predicting the most optimal asparaginase, this technique can also be used to predict the most optimal enzymes where the substrate is known and the structure of one of the homologs is solved.

bioinformatics↗

bioRxiv2020

Evolution and origin of sliding clamp in bacteria, archaea and eukarya

Replication of DNA is an essential process in all domains of life. A protein often involved without exception in replication is the sliding clamp. The sliding clamp encircles the DNA and helps replicative polymerase stay attached to the replication machinery increasing the processivity of the polymerase. In eukaryotes and archaea the sliding clamp is called the Proliferating Cell Nuclear Antigen (PCNA) and consists of two domains. This PCNA forms a trimer encircling the DNA as a hexamer. In bacteria, the structure of the sliding clamp is highly conserved, but the protein itself, called beta clamp, contains three domains, which dimerize to form a hexamer. The bulk of literature touts a conservation of the structure of the sliding clamp, but fails to recognize conservation of protein sequence among sliding clamps. In this paper we have used PSI blast to the second interation in NCBI to show a statistically significant sequence homology between Pyrococcus furiosus PCNA and Kallipyga gabonensis beta clamp. The last two domains of beta clamp align with the two domains of PCNA. This homology data demonstrates that PCNA and beta clamp arose from a common ancestor. In this paper, we have further used beta clamp and PCNA sequences from diverse bacteria, archaea and eukarya to build maximum likelihood phylogenetic tree. Most, but not all, species in different domains of life harbor one sliding clamp from vertical inheritance. Some of these species that have two or more sliding clamps have acquired them from gene duplication or horizontal gene transfer events.

molecular biology↗