Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein

Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.

The attachment of Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase

ABSTRACTReverse transcriptases, RTs, are a standard tool in both fundamental studies and diagnostics used for transcriptome profiling, virus RNA testing and other tasks. RTs should possess elevated temperature optimum, high thermal stability, processivity, and tolerance to contaminants originating from the biological substances under analysis or the purification reagents. Here, we have constructed a set of chimeric RTs, based on the combination of MuLV-RT and DNA-binding domains: the DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein, Sso7d counterpart, from Sulfolobus tokodaii. Chimeric RTs showed the same optimal temperature and the efficacy of terminal transferase reaction as the original M-MuLV RT. Processivity and the efficiency in cDNA synthesis of the chimeric RT with Sto7d at C-end were increased several-fold. The attachment of Sto7d enhanced the M-MuLV RT tolerance to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood, and human blood plasma. Thus, fusing M-MuLV RT with an additional domain resulted in more robust and efficient RTs.

Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA

SummaryReplicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography and protein-protein interaction measurements - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA with an unprecedented level of detail. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation phase and a processive phase during replication.

Mechanism of electroneutral sodium/proton antiporter from transition-path shooting

Na+/H+ antiporters exchange sodium ions (Na+) and protons (H+) on opposite sides of lipid membranes, using the gradient of one ion to drive the uphill transport of the other. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP in continuous and unbiased molecular dynamics trajectories that cover the entire transport cycle. We overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations by transition-path shooting. In this way, we selectively capture the rare events in which the six-helix-bundle transporter domain spontaneously moves up and down to shuttle protons and ions across the membrane. The simulations reveal two hydrophobic gates above and below the ion-binding sites that open and close in response to the bundle motion. Weakening the outside gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.