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.