Biology subjects

Dorawa, S.

Publications and source records attributed to Dorawa, S..

3 recordsLinked to original sources

bioRxiv2026

Hot Pursuit: Bioinformatic and Biochemical Characterization of a Hyperthermophilic Family B DNA Polymerase from Pyrolobus fumarii A1

Thermostable DNA polymerases are indispensable tools in molecular biology, yet enzymes from the most extreme hyperthermophiles remain largely uncharacterized. Here, we report the biochemical and structural characterization of a family B DNA polymerase from Pyrolobus fumarii A1 (Pyrfu pol), one of the most thermoresistant archaea described to date. The enzyme was efficiently overproduced in E. coli Rosetta 2(DE3)[pLysS] and purified to homogeneity using a two-step protocol that combined heat treatment with immobilized metal affinity chromatography (IMAC). Bioinformatic analysis confirmed the canonical family B architecture, while AlphaFold-based structural modeling and comparative analysis with mesophilic RB69 DNA polymerase revealed a well-conserved structural core alongside thermoadaptive features. Radiolabel incorporation assays demonstrated enzymatic activity over a broad ionic strength range and an absolute requirement for Mg2{square}. PCR-based optimization confirmed these findings and revealed broad pH tolerance (6.5-11.0). Notably, Tris inhibited radiolabel-based assays (pH 7.0) yet proved essential for efficient PCR amplification (pH 8.5), suggesting a context-dependent role of buffer composition in polymerase activity. Processivity assays confirmed amplification of DNA fragments up to approximately 8,000 bp. Replication fidelity, assessed by the lacZ-based assay, showed a 2.9-fold improvement over Taq polymerase. Urea-nanoDSF yielded an exceptional melting temperature of 105.9 {+/-} 0.08 {degrees}C. Pyrfu pol also demonstrated tolerance to common PCR inhibitors, highlighting its potential utility in molecular biology applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/734501v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a94cb0org.highwire.dtl.DTLVardef@1e9e1b9org.highwire.dtl.DTLVardef@1e240d8org.highwire.dtl.DTLVardef@10f47b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Beyond thermal unfolding: urea-gradient nanoDSF approach for thermostability analysis of kinetically stable hyperthermophilic proteins

In this study, we demonstrate that urea enables reliable melting temperature (Tm) determination of hyperthermostable proteins by nano differential scanning fluorimetry (nanoDSF) Under native conditions, Pfu DNA polymerase and its Sso7d-fusion variant showed no detectable unfolding transitions, despite their Tm values falling within the instruments operational range, reflecting their extreme kinetic stability. In the presence of up to 7 M urea, intrinsic tyrosine and tryptophan fluorescence revealed clear unfolding transitions, yielding extrapolated Tm values of 104.8 {+/-} 0.09 {degrees}C for Pfu and 106.8 {+/-} 0.33 {degrees}C for its Sso7d-fusion variant. These results demonstrate that urea-gradient nanoDSF overcomes both instrumental and kinetic limitations, providing a simple and robust method for assessing the thermal stability of (hyper)thermostable proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/717478v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@960d9org.highwire.dtl.DTLVardef@1b5613forg.highwire.dtl.DTLVardef@1039a08org.highwire.dtl.DTLVardef@1759841_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system

Deep-sea hydrothermal vent systems with prevailing extreme thermal conditions for life offer unique habitats to source heat tolearant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid- Ocean Ridges. By sequence comparisons against the MEROPS-MPRO database, globupain showed highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the active zymogen and 13 mutant substitution variants allowed assesment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca{superscript 2}. When activated, the 52 kDa proenzyme was processed at Lys137 and Lys144 into a 12 kDa light- and 32 kDa heavy chain heterodimer. A structurally conserved His132/Cys185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR- aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51 {+/-} 0.09{degrees}C) with optimal activity at 75 {degrees}C and pH 7.1. By characterizing globupain, our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases have been expanded. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

microbiology↗