Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.     

Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains

Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.     

A Comparative Study on Torque Requirement During Extrusion Pretreatment of Different Feedstocks

Extrusion pretreatment of biomass can be one of the viable continuous pretreatment methods. The torque requirement of feedstock during extrusion was an important factor, and it was not reported in the literature. Screw compression ratio, screw speed, barrel temperature, and feedstock moisture content are the contributing factors to the torque. The current study was undertaken to investigate the effect of screw compression ratio, screw speed, temperature on torque requirement for different moisture content of switchgrass, prairie cord grass, corn stover, and big bluestem and to compare the torque requirement among the selected feedstocks. Biomass was extruded in a lab scale single-screw extruder with different screw compression ratios (2:1 and 3:1), screw speeds (50, 100, and 150?rpm), and barrel temperatures (50°C, 100°C, and 150°C) over a range of moisture contents (15%, 25%, 35%, and 45% wb). Statistical analyses revealed that all the independent variables considered in this study had a significant effect on torque requirement for the selected feedstocks. Among the independent variables considered moisture content, screw speed, and temperature had a negative effect on torque requirement for all the feedstocks. Switchgrass required the highest torque followed by corn stover, big bluestem, and prairie cord grass.     

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Regulation of minichromosome maintenance helicase activity by Cdc6

Genetic studies, together with amino acid and structural similarities to the clamp loaders of DNA polymerase sliding clamps, have suggested that the Cdc6 protein may function as a loader for the eukaryotic replicative helicase, the minichromosome maintenance (MCM) complex. Thus, Cdc6 may act as the functional homologue of the bacterial DnaC that utilizes ATP hydrolysis to assemble the DnaB helicase at the origin. This report shows that the helicase activity of an MCM homologue from the archaeon Methanothermobacter thermautotrophicus is inhibited in the presence of the Cdc6 homologues. This inhibitory activity is dependent, as for DnaC, on ATP binding to Cdc6. Moreover, an intact Cdc6 winged helix domain is required for efficient inhibition. Two-hybrid analyses indicated that MCM and Cdc6 interact and that the interaction is mediated by the winged helix domain. Analysis of Cdc6 and MCM homologues from several archaea exhibited differences in the inhibitory activity suggesting divergence in function in Cdc6 and MCM homologues among the archaea.     

The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover,Cellulase Structure,and Adsorption

Enzymatic hydrolysis of lignocellulosic biomass is limited by rapid cellulase deactivation, consequently requiring large amounts of enzyme to maintain acceptable biomass conversion. In this study, a new approach to improve lignocellulose hydrolysis was investigated. Performing enzymatic hydrolysis of corn stover (CS) in the presence of polymeric–surfactant micelles (PMs) was demonstrated to improve hydrolysis yield to a greater extent than using only surfactant micelles. Application of 2 % (w/w) of polyethylene glycol (PEG 6000) with casein, Tween-20, and Triton X-100 at levels above the critical micelle concentrations increased the hydrolysis yield of CS containing high-bound lignin (extrusion-pretreated) by up to 87.8, 11.7, and 7.5 %, respectively. These PMs were not effective during enzymatic hydrolysis of biomass lacking lignin (Avicel) or alkali-pretreated CS (7.2 % lignin). The main reasons for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween, and PEG-Triton were enhanced cellulase solubilization; reformation of α-helix substructure; and combination of induced cellulase solubilization, α-helix reformation, and chemical changes in the microstructure of biomass, respectively. Deformation of the cellulase substructure during hydrolysis of biomass and its subsequent reformation in the presence of surfactants were shown in this study for the first time. Chemical changes in the microstructure of biomass (e.g., lignin side changes, C–O bonds, and amorphous cellulose) were found to be another potential reason for the effectiveness of surfactants when they are incubated at above 6 g/L for 72 h with biomass.     

DNA binding by the Methanothermobacter thermautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase

The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.