Crystal structures of the molecular class A β-lactamase TEM-171 and its complexes with tazobactam.

The resistance of bacteria to β-lactam antibiotics is primarily caused by the production of β-lactamases. Here, novel crystal structures of the native β-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder.

Modulation of α-Chymotrypsin Conjugated to Magnetic Nanoparticles by the Non-Heating Low-Frequency Magnetic Field: Molecular Dynamics, Reaction Kinetics, and Spectroscopy Analysis.

Enzymes conjugated to magnetic nanoparticles (MNPs) undergo changes in the catalytic activity of the non-heating low-frequency magnetic field (LFMF). We apply in silico simulations by molecular dynamics (MD) and in vitro spectroscopic analysis of the enzyme kinetics and secondary structure to study α-chymotrypsin (CT) conjugated to gold-coated iron oxide MNPs. The latter are functionalized by either carboxylic or amino group moieties to vary the points of enzyme attachment.

Molecular dynamics simulations of two GH74 endo-processive xyloglucanases and the mutated variants to understand better the mechanism of the enzyme action.

Background: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis.

Methods: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74) and Myceliophthora thermophila (MtXeg74A) were carried out.

The Role of the Ω-Loop in Regulation of the Catalytic Activity of TEM-Type β-Lactamases.

Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed β-lactamases (βLs). The evolution of serine-class A β-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme's active site.

In Situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field.

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field.

Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type β-lactamases.

Highly mutable β-lactamases are responsible for the ability of Gram-negative bacteria to resist β-lactam antibiotics. Using site-directed mutagenesis technique, we have produced a number of recombinant analogs of naturally occurring TEM-type β-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) β-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β-lactamases.

Thermodynamics and molecular insight in guest-host complexes of fluoroquinolones with β-cyclodextrin derivatives, as revealed by ATR-FTIR spectroscopy and molecular modeling experiments.

β-Сyclodextrin (CD) is a perspective class of excipients used in pharmaceutical formulations to enhance solubility, bioavailability, and pharmacokinetics of various poorly soluble drugs, forming a non-covalent guest-host complex. However, the development of such formulations is usually a very laborious and time-consuming process due to lack of appropriate analytical tools to directly track and study the detailed molecular mechanism of such complex formation. Here, using guest-host complexes of fluoroquinolones (FQ) with CDs, as an example, we demonstrate the utility of ATR-FTIR to determine the thermodynamic stability, as well as structural features associated with complex formation, including involvement of certain functional groups.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity.

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Background: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.

Methods/principal Findings: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs.

Introducing DInaMo: A Package for Calculating Protein Circular Dichroism Using Classical Electromagnetic Theory.

The dipole interaction model is a classical electromagnetic theory for calculating circular dichroism (CD) resulting from the π-π* transitions of amides. The theoretical model, pioneered by J. Applequist, is assembled into a package, DInaMo, written in Fortran allowing for treatment of proteins.