Engineering an allosteric binding site for aminoglycosides into TEM1-β-Lactamase.

Allosteric regulation of enzyme activity is a remarkable property of many biological catalysts. Up till now, engineering an allosteric regulation into native, unregulated enzymes has been achieved by the creation of hybrid proteins in which a natural receptor, whose conformation is controlled by ligand binding, is inserted into an enzyme structure. Here, we describe a monomeric enzyme, TEM1-β-lactamase, that features an allosteric aminoglycoside binding site created de novo by directed-evolution methods.

Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase.

In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors.

Engineering allosteric regulation into biological catalysts.

Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site.

Pulmonary arterial hypertension in a patient with stage II sarcoidosis and Hashitoxicosis.

Although pulmonary arterial hypertension is usually associated with advanced stages of sarcoidosis, its occurrence in early stage disease is rare. Herein, a case of associated pulmonary arterial hypertension in the setting of Hashitoxicosis and stage II pulmonary sarcoidosis is reported. The case of associated pulmonary arterial hypertension occurred in a young female without clinically significant medical history and who completely recovered after receiving oral corticotherapy only.

Structure of PBP-A from Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A beta-lactamases.

Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, D-alanyl-D-alanine peptidases and beta-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while beta-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit d-alanyl-d-alanine peptidases by forming stable covalent penicilloyl enzymes.

A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases.

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed.

Complete disappearance of lung abnormalities on high-resolution computed tomography: a case of histiocytosis X.

A case of pulmonary Langerhans cell histiocytosis, proved by both lung high-resolution computed tomography and lung biopsy, is described. Following smoking cessation, lung nodules and cysts gradually disappeared on serial computed tomography scans, with complete clearance of the lesions after 12 months. The role of tobacco smoking is discussed, in detail, against the background of the literature.

Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops.

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants.

Selection of allosteric beta-lactamase mutants featuring an activity regulation by transition metal ions.

Libraries of phage-displayed beta-lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild-type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double-selection protocol was applied: The phage-displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal-specific manner.

Production in Saccharomycescerevisiae of MS2 virus-like particles packaging functional heterologous mRNAs.

Recently, DNA bacteriophages (M13, lambda) have been genetically engineered to transfer genes into mammalian cells. Although efficiencies observed are still relatively low, this opens the possibility of using these viruses as a new class of transfection agents not only for fundamental research purposes but also in gene therapy protocols or in other applications like vaccination. In this respect, it has been shown that a lambda bacteriophage engineered to express the hepatitis B surface antigen in mammalian cells could elicit an immune response against this antigen in mice and rabbits without any specific targeting of the bacteriophage.

Engineering of non-natural receptors.

Rational design, usually guided by computational prediction, and selection from libraries of variants of natural proteins have been used with success in the engineering of novel non-natural receptors. Many of these engineered protein binders will find use in biotechnological, diagnostic and medical applications, sometimes in the place of natural antibodies.

Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid.

Phage display has evolved during the past 15 years as a powerful technique to select, from libraries of peptides or proteins, binders for various targets or to evolve new functions in proteins. In recent years, the knowledge acquired in phage display technology was exploited to engineer phages as vehicles for receptor-mediated gene delivery. The first vectors generated provided the proof of the concept that development of gene delivery vehicles based on phages was feasible.

Spontaneous rupture of a Dacron prosthesis.

This case report concerns a spontaneous rupture of a Dacron prosthesis 19 years after its insertion. The rupture occurRed in the mid-graft portion, remote from the anastomoses and was not associated with proved infection. It was the result of an intrinsic deterioration of the graft textile structure.

TEM-1 beta-lactamase as a scaffold for protein recognition and assay.

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated.

Selection of metalloenzymes by catalytic activity using phage display and catalytic elution.

The metallo-beta-lactamase betaLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage-bound enzyme fd-betaLII was shown to be active on benzylpenicillin as substrate; it could be inactivated by complexation of the essential zinc(II) ion with EDTA and reactivated by addition of a zinc(II) salt. A selection process was designed to extract active phage-bound enzymes from libraries of mutants in three steps: 1.

Novel concepts for selection of catalytic activity.

The selection of mutant enzymes with novel properties from libraries is emerging as a very powerful strategy for enzyme engineering. The past year has witnessed significant progress on several fronts: new and improved methods have been developed for the creation of libraries and advances have been made in screening and selection techniques. The results achieved demonstrate the enormous potential of the methods and leave questions open for further studies.

CENTA as a chromogenic substrate for studying beta-lactamases.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

[Anatomic and clinical features of a case of synoviosarcoma].

An asymptomatic patient presented a pulmonary metastatis of a synovial sarcoma 15 years after resection of the primary tumor localized in the right groin area. The pathology findings demonstrated the non-specific variable nature of the biphasic epithelial and mesenchymatous feature of such tumors. No treatment was initiated and the patient has remained asymptomatic after five years follow-up.