Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways.

Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC.

Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity.

Enhanced activity against a third-generation cephalosporin by destabilization of the active site of a class A beta-lactamase.

The β-lactamase BlaC conveys resistance to a broad spectrum of β-lactam antibiotics to its host Mycobacterium tuberculosis but poorly hydrolyzes third-generation cephalosporins, such as ceftazidime. Variants of other β-lactamases have been reported to gain activity against ceftazidime at the cost of the native activity. To understand this trade-off, laboratory evolution was performed, screening for enhanced ceftazidime activity.

Asp179 in the class A β-lactamase from Mycobacterium tuberculosis is a conserved yet not essential residue due to epistasis.

Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the β-lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins.

The roles of highly conserved, non-catalytic residues in class A β-lactamases.

Evolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non-catalytic residues, 11% of all residues, in class A β-lactamase were analyzed by studying the effect of 146 mutations on in cell and in vitro activity, folding, structure, and stability. Residues around the catalytic residues (second shell) contribute to fine-tuning of the active site structure.

The G132S Mutation Enhances the Resistance of β-Lactamase against Sulbactam.

The current rise of antibiotic resistant forms of is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam.

Two β-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics.

The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties.

Conserved residues Glu37 and Trp229 play an essential role in protein folding of β-lactamase.

Evolutionary robustness requires that the number of highly conserved amino acid residues in proteins is minimized. In enzymes, such conservation is observed for catalytic residues but also for some residues in the second shell or even further from the active site. β-Lactamases evolve in response to changing antibiotic selection pressures and are thus expected to be evolutionarily robust, with a limited number of highly conserved amino acid residues.

Modification of the catalytic subunit of plasma fibrin-stabilizing factor under induced oxidation.

For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the β-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the β-barrel 1 (Met596), and the β-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.