Bacterium Thermus Thermophilus Research Articles

Crystal structure of guanosine 5'-monophosphate synthetase from the thermophilic bacterium Thermus thermophilus HB8.

Guanosine 5'-monophosphate (GMP) synthetase (GuaA) catalyzes the last step of GMP synthesis in the purine nucleotide biosynthetic pathway. This enzyme catalyzes a reaction in which xanthine 5'-monophosphate (XMP) is converted to GMP in the presence of Gln and ATP through an adenyl-XMP intermediate. A structure of an XMP-bound form of GuaA from the domain Bacteria has not yet been determined. In this study, the crystal structure of an XMP-bound form of GuaA from the thermophilic bacterium Thermus thermophilus HB8 (TtGuaA) was determined at a resolution of 2.20 Å and that of an apo form of TtGuaA wasdetermined at 2.10 Å resolution. TtGuaA forms a homodimer, and the monomer is composed of three domains, which is a typical structure for GuaA. Disordered regions in the crystal structure were obtained from the AlphaFold2-predicted model structure, and a model with substrates (Gln, XMP and ATP) was constructed for molecular-dynamics (MD) simulations. The structural fluctuations of the TtGuaA dimer as well as the interactions between the active-site residues were analyzed by MD simulations.

Identification of subcomplexes and protein-protein interactions in the DNA transporter of Thermus thermophilus HB27

The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10−5 bp−1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.

In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here. Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC3(5)). The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Expression of a recombinant protein by an acetic acid bacterial host

We evaluated the suitability of Komagataeibacter europaeus, a vinegar production organism adept at synthetic media growth, as a host for heterologous gene expression. Cryptic plasmids (pGE1 and pGE2 derivatives) from K. europaeus strain KGMA0119 were employed as vectors for heterologous gene expression. The focus was placed on the groES promoter as a potential inducible switch. The groES promoter was fused with the EGFP gene and introduced into a pGE1 derivative to assess its suitability. Ethanol, acetic acid, and heat stresses were examined under various conditions for induction. EGFP transcription surged 600-fold when late logarithmic phase K. europaeus cells, cultured at 30 °C, endured heat stress at 40 °C, coupled with 20% acetic acid and 30% ethanol stress after an additional 6-hour cultivation. This robust induction system was then applied to express two proteins, Tth pol from the thermophilic bacterium Thermus thermophilus strain M1 and UPV230, a restriction enzyme from the acid-tolerant microorganism Ureaplasma parvum, known to cause vaginal infections and miscarriages. Both Tth pol and UPV230 were successfully expressed in K. europaeus cells and purified. The recovery of Tth pol from K. europaeus cells (480 µg protein per liter culture) was approximately half that from E. coli (960 µg protein per liter culture). In contrast, UPV230 recovery from K. europaeus cells (640 µg protein per liter culture) was nearly 10 times higher than that from Escherichia coli (66 µg protein per liter). The data highlights the potential of acetic acid bacteria as a host for producing acidophilic proteins. The shift in recognition from a 6-base sequence to a 4-base sequence of UPV230 was observed, accompanied by a change in structure as the pH transitioned from acidic pH to near-neutral pH.

Approach enhancing nitrate removal from actual municipal wastewater by integrating electric-magnetic field with Fe0 in UMSR reactor

This study aimed to investigate the effectiveness of combining zero-valent iron (Fe0) with an electric-magnetic field (EMF) with an intensity of 48-mT generated by an alternating current (AC) of 6 V and 50 Hz applied deep within a reactor using actual wastewater obtained from a secondary stage of a wastewater treatment plant (WWTP). The lab-scale experiment was conducted using the UMSR reactor, maintaining a controlled temperature of 25 ± 2 °C and a hydraulic retention time (HRT) of 5-hour, with a dissolved oxygen (DO) level below 1.5 mg/L. Fine Fe0 particles with a size of ≤ 0.3 µm were added into the reactor at a concentration of 3 g/L. The actual wastewater had an approximate nitrate nitrogen (NO3–-N) concentration of 30 ± 2 mg/L, and the substrate mass ratio of carbon to nitrogen (C:N) was at 1:1. The results examined the performance of the EMF-Fe0 system in terms of nitrate removal efficiency and the enrichment of denitrifying bacteria, achieving a nitrate removal rate of 67 % with an effluent NO3–-N concentration of 10 mg/L and a chemical oxygen demand (COD) concentration of 11.8 mg/L at removal rate of 60 %. Illumina sequencing revealed various enriched denitrifying bacteria after coupling EMF with Fe0, including autotrophic Nitrospira (NOB) at 4 %, denitrification bacteria like Hydrogenophaga at 1.3 %, Hyphomicrobium at 1 %, and Sphingobium at 5 %, along with thermophilic bacteria Thermus at 1.1 % and Meiothermus at 3.5 %. PICRUSt predicted bacterial functions and analyzed interactions, while KEGG and GO analyses linked nitrogen removal pathways to specific species, genes, and enzymes in the EMF-Fe0 system.

Crystal structure of adenylosuccinate lyase from the thermophilic bacterium Thermus thermophilus HB8.

Adenylosuccinate lyase (PurB) catalyzes two distinct reactions in the purine nucleotide biosynthetic pathway using the same active site. The ability to recognize two different sets of substrates is of structural and evolutionary interest. In the present study, the crystal structure of PurB from the thermophilic bacterium Thermus thermophilus HB8 (TtPurB) was determined at a resolution of 2.38 Å by molecular replacement using a structure predicted by AlphaFold2 as a template. The asymmetric unit of the TtPurB crystal contained two TtPurB molecules, and some regions were disordered in the crystal structure. The disordered regions were the substrate-binding site and domain 3. TtPurB forms a homotetramer and the monomer is composed of three domains (domains 1, 2 and 3), which is a typical structure for the aspartase/fumarase superfamily. Molecular dynamics simulations with and without substrate/product were performed using a full-length model of TtPurB which was obtained before deletion of the disordered regions. The substrates and products were bound to the model structures during the MD simulations. The fluctuations of amino-acid residues were greater in the disordered regions and became smaller upon the binding of substrate or product. These results demonstrate that the full-length model obtained using AlphaFold2 can be used to generate the coordinates of disordered regions within the crystal structure.

Modeling the role of charged residues in thermophilic proteins by rotamer and dynamic cross correlation analysis.

Discerning the determinants of protein thermostability is very important both from the theoretical and applied perspective. Different lines of evidence seem to indicate that a dynamical network of salt bridges/charged residues plays a fundamental role in the thermostability of enzymes. In this work, we applied measures of dynamic variance, like the Gini coefficients, Kullback-Leibler (KL) divergence and dynamic cross correlation (DCC) coefficients to compare the behavior of 3 pairs of homologous proteins from the thermophilic bacterium Thermus thermophilus and mesophilic Escherichia coli. Molecular dynamic (MD) simulations of these proteins were performed at 303K and 363K. In the characterization of their side chain rotamer distributions, the corresponding Gini coefficients and KL-divergence both revealed significant correlations with temperature. Similarly, a DCC analysis revealed a higher trend to de-correlate the movement of charged residues at higher temperatures in the thermophilic proteins, when compared with their mesophilic homologues. These results highlight the importance of dynamic electrostatic network interactions for the thermostability of enzymes.

The parABSm system is involved in megaplasmid partitioning and genome integrity maintenance in Thermus thermophilus.

The characteristics of the parABS system in polyploid bacteria are barely understood. We initially analyzed the physiological functions and mechanisms of the megaplasmid parABSm system in the thermophilic polyploid bacterium Thermus thermophilus. Deletion of parABm was possible only when a plasmid-born copy of parABm was provided, indicating that these genes are conditionally essential. The cell morphology of the parABm deletion mutant (ΔparABm) was changed to some extent, and in certain extra-large or twisted cells, the nucleoids were dispersed and damaged. Compared with that of the wild type, the frequency of anucleate cells was significantly increased. Genome content analyses showed that ΔparABm had lost ∼160 kb of megaplasmid and ∼23 kb of chromosomal sequences, respectively. Genome fluorescent tagging and PFGE experiments demonstrated that the truncated megaplasmid was frequently interlinked and could not be segregated correctly; thus, certain daughter cells eventually lost the entire megaplasmid and became twisted or enlarged with damaged nucleoids. Further, we found that when the megaplasmid was lost in these cells, the toxins encoded by the megaplasmid toxin-antitoxin (TA) systems (VapBC64_65 and VapBC142_143) would exert detrimental effects, such as to fragment DNA. Thus, parABSm might ensure the existence of these TA systems, thereby preventing genomic degradation. Together, our results suggested that in T. thermophilus, the megaplasmid-encoded parABS system plays an essential role in the megaplasmid partitioning process; also it is an important determination factor for the genome integrity maintenance.

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes.

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.

An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory

Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the application of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.

Structural and thermodynamic insights into a novel Mg2+-citrate-binding protein from the ABC transporter superfamily.

More than one third of proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria they are translocated by Na+/citrate symporters. In this study, the presence of a novel divalent-metal-ion-complexed citrate-uptake system that belongs to the primary active ABC transporter superfamily is reported. For uptake, the metal-ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains for their transport. This study reports crystal structures of an Mg2+-citrate-binding protein (MctA) from the Gram-negative thermophilic bacterium Thermus thermophilus HB8 in both apo and holo forms in the resolution range 1.63-2.50 Å. Despite binding various divalent metal ions, MctA possesses the coordination geometry to bind its physiological metal ion, Mg2+. The results also suggest an extended subclassification of cluster D SBPs, which are known to bind and transport divalent-metal-ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant MctA proteins suggests a gating mechanism of ligand entry following an `asymmetric domain movement' of the N-terminal domain for substrate binding.

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

Comparison of Spatial Structures and Packaging of Phosphorybosil Pyrophosphate Synthetase 2 from Thermus thermophilus HB27 in Rhombohedral and Tetragonal Crystals

We report the spatial structure of phosphoribosyl pyrophosphate synthetase 2 from the thermophilic bacterium Thermus thermophilus HB27 (TthPRPPS2) obtained at a 1.85 Å resolution using a diffraction set collected from rhombohedral crystals (space group R32-h), grown with lithium sulfate as a precipitant. This crystal structure was compared with the structure of TthPRPPS2, previously obtained at a 2.2 Å resolution using diffraction sets from the tetragonal crystals (space group P41212), grown with ammonium sulfate as a precipitant. The comparison of these structures allows the study of the differences between protein molecules in both crystalline structures, as well as the packaging of enzyme molecules in crystals of both spatial groups. Our results may contribute to the research of the structural basis of catalytic activity and substrate specificity of this enzyme.

Comparative study on tertiary contacts and folding of RNase P RNAs from a psychrophilic, a mesophilic/radiation-resistant, and a thermophilic bacterium

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8–P4 and L18–P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8–P4 and L18–P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9–P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.

The β-hairpin from the Thermus thermophilus HB27 laccase works as a pH-dependent switch to regulate laccase activity

The multi-copper oxidase from the hyper-thermophilic bacteria Thermus thermophilus (Tth-MCO), has been previously characterized and described as an example of a laccase with low catalytic properties, especially when it is compared with the activity of fungal laccases, but it is active at high temperatures. Structurally, Tth-MCO has a unique feature: a β-hairpin near the T1Cu site, which is not present in any other laccases deposited at the PDB. This β-hairpin has an expected crystallographic behavior in solvent-exposed areas of a crystallized protein: lack of electron density, high B-values and several crystalline contacts with neighboring crystallographic copies; however, its dynamical behavior in solution and its biological implications have not been described. Here, we describe four new Tth-MCO crystallographic structures, and the β-hairpin behavior has been analyzed by molecular dynamics simulations, considering the effect of pH and temperature. The β-hairpin new crystallographic conformations described here, together with their dynamics, were used to understand the pH-restrained laccase activity of Tth-MCO against substrates as syringaldazine. Remarkably, there are insertions in laccases from Thermus and Meiothermus genus, sharing the same position and a methionine-rich composition of the Tth-MCO β-hairpin. This unique high methionine content of the Tth-MCO β-hairpin is responsible to coordinate, Ag+1 and Hg+1 in oxidative conditions, but Cu+1 and Cu+2 are not coordinated in crystallographic experiments, regardless of the redox conditions; however, Ag+1 addition does not affect Tth-MCO laccase activity against syringaldazine. Here, we propose that the pH-dependent β-hairpin dynamical behavior could explain, at least in part, the inefficient laccase activity displayed by Tth-MCO in acidic pH values.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.