Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 3_(10) helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity

Chromosome segregation in Archaea : SegA– and SegB–DNA complex structures provide insights into segrosome assembly

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation

Mutations Altering the Interplay between GkDnaC Helicase and DNA Reveal an Insight into Helicase Unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2–4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction

NrdR overexpression influences changes in bacterial morphology.

<p><b>(A)</b> Colony morphology of WT, ΔNrdR and OE-NrdR <i>E</i>. <i>coli</i> strains grown on LB medium. <b>(B)</b> Magnification (×100) of <i>E</i>. <i>coli</i> strains. Overexpression of NrdR resulted in bacterial aggregates (red arrows) and coccobacilli (short rods; green arrows). <b>(C)</b> Transmission electron microscopy of the WT, ΔNrdR and OE-NrdR <i>E</i>. <i>coli</i> strains at high magnification (magnification, ×21,000). Differences in flagella and cell walls among the <i>E</i>. <i>coli</i> strains can be observed.</p

Heat maps representing differential regulation of proteins in <i>E</i>. <i>coli</i> following NrdR-deletion and -overexpression.

<p><b>(A)</b> Protein categories upregulated in the ΔNrdR strain but downregulated in the OE-NrdR strain. <b>(B)</b> Protein categories upregulated in the ΔNrdR strain but with no change for the OE-NrdR strain. The color scale indicates differential regulation of protein amounts relative to WT <i>E</i>. <i>coli</i> control, with upregulation indicated by green shading and downregulation by red. Genes are listed alphabetically.</p

Proteomic response of <i>E</i>. <i>coli</i> to NrdR-deletion and -overexpression.

<p><b>(A)</b> LC-MS/MS resulted in identification of 818 soluble proteins of sufficient abundance to evaluate across strains. TheΔNrdR strain had more upregulated proteins compared to the wild-type, while the OE-NrdR strain had a larger percentage of downregulated proteins. The number of proteins in each category is indicated. <b>(B)</b> Selective distribution of upregulated and downregulated proteins according to log₂-fold change in the ΔNrdR (left panel) and OE-NrdR (right panel) mutants. X-axis represents the number of genes and Y-axis represents ranges of n-fold changes in protein expression.</p