Domain Mobility in the ORF2p Complex Revealed by Molecular Dynamics Simulations and Big Data Analysis.

ORF2p (open reading frame 2 protein) is a multifunctional multidomain enzyme that demonstrates both reverse transcriptase and endonuclease activities and is associated with the pathophysiology of cancer. The 3D structure of the entire seven-domain ORF2p complex was revealed with the recent achievements in structural studies. The different arrangements of the CTD (carboxy-terminal domain) and tower domains were identified as the "closed-ring" and "open-ring" conformations, which differed by the hairpin position of the tower domain, but the structural diversity of these complexes has the potential to be more extensive.

Molecular Basis of the Substrate Specificity of Phosphotriesterase from : A Combined QM/MM MD and Electron Density Study.

The occurrence of organophosphorus compounds, pesticides, and flame-retardants in wastes is an emerging ecological problem. Bacterial phosphotriesterases are capable of hydrolyzing some of them. We utilize modern molecular modeling tools to study the hydrolysis mechanism of organophosphorus compounds with good and poor leaving groups by phosphotriesterase from Pseudomonas diminuta (Pd-PTE).

Quantum-based Modeling of Protein-ligand Interaction: The Complex of RutA with Uracil and Molecular Oxygen.

Modern quantum-based methods are employed to model interaction of the flavin-dependent enzyme RutA with the uracil and oxygen molecules. This complex presents the structure of reactants for the chain of chemical reactions of monooxygenation in the enzyme active site, which is important in drug metabolism. In this case, application of quantum-based approaches is an essential issue, unlike conventional modeling of protein-ligand interaction with force fields using molecular mechanics and classical molecular dynamics methods.

Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase.

We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to the reaction products, cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi), catalyzed by ACs initiated by excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes that modulate cellular concentrations of signaling messengers. The photoactivated AC from the soil bacterium sp.

Proof of concept for poor inhibitor binding and efficient formation of covalent adducts of KRAS and ARS compounds.

The use of selective covalent inhibitors with low binding affinity and high reactivity with the target enzyme is a promising way to solve a long-standing problem of the "undruggable" RAS-like proteins. Specifically, compounds of the ARS family that prevent the activation of the GDP-bound G12C mutant of Kirsten RAS (KRAS) are in the focus of recent experimental research. We report the first computational characterization of the entire reaction mechanism of the covalent binding of ARS-853 to the KRASG12C·GDP complex.

Competition between two cysteines in covalent binding of biliverdin to phytochrome domains.

In this work, we disclose a mechanism of competing chemical reactions of protein assembly for a bacterial phytochrome using modern methods of molecular modeling. The recently designed variant of a near-infrared fluorescent protein miRFP670 shows novel and unexpected features of covalent binding of the biliverdin chromophore to cysteine residues in the phytochrome domains GAF and PAS. Upon protein assembly, biliverdin reacts either with a cysteine from GAF, or with two cysteines, one from GAF and one from PAS.

Tracking the fate of microbially sequestered carbon dioxide in soil organic matter.

The microbial contribution to soil organic matter (SOM) has recently been shown to be much larger than previously thought and thus its role in carbon sequestration may also be underestimated. In this study we employ (13)C ((13)CO₂) to assess the potential CO₂ sequestration capacity of soil chemoautotrophic bacteria and combine nuclear magnetic resonance (NMR) with stable isotope probing (SIP), techniques that independently make use of the isotopic enrichment of soil microbial biomass. In this way molecular information generated from NMR is linked with identification of microbes responsible for carbon capture.

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis.

The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F.

The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor.

Expression of the phosphonoalanine-degradative gene cluster from Variovorax sp. Pal2 is induced by growth on phosphonoalanine and phosphonopyruvate.

The phosphonopyruvate hydrolase (PalA) found in Variovorax sp., Pal2, is a novel carbon-phosphorus bond cleavage enzyme, which is expressed even in the presence of high levels of phosphate, thus permitting phosphonopyruvate to be used as the sole carbon and energy source. Analysis of the regions adjacent to the palA gene revealed the presence of the five structural genes that constitute the 2-amino-3-phosphonopropionic acid (phosphonoalanine)-degradative operon.

Potential for phosphonoacetate utilization by marine bacteria in temperate coastal waters.

Phosphonates are organic compounds that contain a C-P bond and are a poorly characterized component of the marine phosphorus cycle. They may represent a potential source of bioavailable phosphorus, particularly in oligotrophic conditions. This study has investigated the distribution of the phnA gene which encodes phosphonoacetate hydrolase, the enzyme that mineralizes phosphonoacetate.

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling.

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: new insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily.

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity.

In vitro detection and characterisation of a polyphosphate synthesising activity in the yeast Candida humicola G-1.

An in vitro detectable polyphosphate-synthesising activity was characterised using two independent assay systems in extracts of the yeast Candida humicola G-1. Its properties were similar to those of a range of bacterial polyphosphate kinase enzymes. PCR amplification of C.

The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp Pal2.

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell.

Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains.

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases.

The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064.

The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase.