S31: Directed evolution of Enterococcus aminoglycoside phosphotransferase type III variants that confer resistance to amikacin

We seek to understand how enzymes adapt to new substrates.  The study of any biological process is facilitated by the adoption of a model system.  The TEM-1 beta-lactamase has served as a model enzyme for directed evolution studies.  It imparts modest resistance upon gram negative bacteria to certain cephalosporins, thereby enabling the artificial evolution of variants that confer enhanced resistance to these antibiotics.  We don't know, however, whether conclusions from these studies can be generalized to other enzymes.  We have chosen the broad spectrum Enterococcus aminoglycoside phosphotransferase type III, or APH(3')-IIIa, as a second model.  APH(3')-IIIa is slightly smaller than TEM-1 beta-lactamase, and is also a monomer.  Unlike TEM-1, however, APH(3')-IIIa is not secreted and  it confers modest resistance to a structurally more diverse set of aminoglycoside antibiotics.  In its wild type form, APH(3')-IIIa  confers modest resistance to the aminoglycoside amikacin, a clinically relevant antibiotic.  Here we describe a proof-of-principle experiment, using a combination of random mutagenesis and recombination to direct the evolution of APH(3')-IIIa variants that confer greater resistance to amikacin.  We are using the APH(3')-IIIa system to verify current models of adaptation, and develop new evolutionary methods.