Metallo-beta-lactamases challenge antimicrobial therapies by their ability to hydrolyze and inactivate a broad spectrum of beta-lactam antibiotics. The potential of these enzymes to acquire enhanced catalytic efficiency through mutation is of great concern. Here, we explore the potential of computational protein design to predict mutants of the imipenemase IMP-1 that modulate the catalytic efficiency of the enzyme against a range of substrates. Focusing on the four amino acid positions 69, 121, 218, and 262, we carried out a number of design calculations. Two mutant enzymes were predicted: the single mutant S262A and the double mutant F218Y-S262A. Compared to IMP-1, the single mutant (S262A) results in the loss of a hydroxyl group and the double mutant (F218Y-S262A) results in a hydroxyl transfer from position 262 to position 218. The presence of both hydroxyl groups at positions 218 and 262 was tested by examining the mutant F218Y. Kinetic constants of IMP-1, the two computationally designed mutants (S262A and F218Y-S262A), and the hydroxyl addition mutant (F218Y) were determined with seven substrates. Catalytic efficiencies are highest for the enzyme with both hydroxyl groups (F218Y) and lowest for the enzyme lacking both hydroxyl groups (S262A). The catalytic efficiencies of the two enzymes with one hydroxyl group each are intermediate, with the F218Y-S262A double mutant exhibiting enhanced hydrolysis of nitrocefin, cephalothin, and cefotaxime relative to IMP-1.
Submitter: Peter Oelschlaeger
Submission Date: Jan. 9, 2019, 9:23 a.m.
|Number of data points||172|
|Proteins||Metallo-beta-lactamase type 2|
|Assays/Quantities/Protocols||Experimental Assay: Melting Temperature (Tm) ; Experimental Assay: kcat/Km ; Experimental Assay: Km ; Experimental Assay: kcat ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of Km ; Derived Quantity: SD of kcat|
|Libraries||Melting Temperature (Tm) ; Activity data for IMP-1 and variants with different beta-lactams (Table S1)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||Metallo-beta-lactamase type 2||P52699||BLAB_SERMA|