Contribution of Mutant Analysis to the Understanding of Enzyme Catalysis: The Case of Class a Beta-Lactamases

Class A beta-lactamases represent a family of well studied enzymes. They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy. Despite the facts that five structures are known at high resolution and that detailed analyses of enzymes modified by site-directed mutagenesis have been performed, their exact catalytic mechanism remains controversial. This review attempts to summarize and to discuss the many available data

Class A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A -lactamases as versatile scaffolds to design hybrid enzymes (referred to as -lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins.Peer reviewe

The Right-Handed Parallel β-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure.

peer reviewedThe complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol-1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the 1H, 13C and 15N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR

Structural and functional characterization of Solanum tuberosum VDAC36

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum , stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.Peer ReviewedPostprint (author's final draft

The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.Peer reviewe

In silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design

Kinetic Study of Laboratory Mutants of NDM-1 Metallo-beta-Lactamase and the Importance of an Isoleucine at Position 35.

peer reviewedTwo laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of alpha-helix content in the mutants

Purification and Characterization of Trehalase From Acyrthosiphon pisum, a Target for Pest Control.

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs (179)PGGRFRELYYWDTY(192) and (479)QWDFPNAWPP(489), a glycine-rich region (549)GGGGEY(554), and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with K(m) and V(max) values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health

Investigation of structure-stabilizing elements in proteins by ion mobility mass spectrometry and collision-induced unfolding

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