Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs Xiaojun Wang, George Minasov and Brian K. Shoichet* Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine 303 East Chicago Avenue Chicago, IL 60611-3008, USA Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the b-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 ! Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity. q 2002 Elsevier Science Ltd. All rights reserved Keywords: protein stability; TEM-1; b-lactamase; antibiotic resistance; evolution *Corresponding author Introduction As new antibiotics are introduced, drug- inactivating resistance enzymes have co-evolved, broadening their activity to ever more elaborate antibiotics. Does the evolution of enzymes with new substrate spectra have a cost? It is conceivable, for instance, that gaining activity against a new substrate might come at the expense of older sub- strates. From a structural standpoint, creating a more versatile active site could destabilize the enzyme. Unlike the well-packed, stability- conferring cores of proteins, active sites are necessarily poorly packed 1 and enlarging them would aggravate this. Also, active sites are pre- organized to recognize substrates, 2 and this pre- organization appears to introduce strain. In active sites, groups bearing the same formal charge are juxtaposed, 3,4 introducing electrostatic repulsion, and conformational strain is often observed among ligand-binding residues. 5 Consequently, it has been possible to make substitutions in active sites that result in highly stabilized mutant enzymes with reduced activity. 6–9 By the same logic, the increased activity of the mutant resist- ance enzymes might increase active-site strain, reducing enzyme stability. Such trade-offs between enzyme activity and stability would act as a con- straint on the evolution of new resistance enzymes. We turned to gain-of-function mutants of TEM-1 b-lactamase, the predominant source of resistance to penicillins in bacteria. The wild-type (WT) enzyme is an excellent penicillinase but has little activity against third generation cephalosporins, such as ceftazidime (CAZ) or cefotaxime (CTX). Such cephalosporins possess bulky oxyimino side- chains that are thought to be too large for the TEM-1 active site (Figure 1(a)). 10,11 Since the intro- duction of these drugs in 1983, mutant extended spectrum b-lactamase (ESBL) TEM enzymes have been found that confer resistance to these drugs in clinical isolates. There are now more than 50 ESBL TEM mutants and more than 18 ESBL mutants of a related SHV b -lactamase†; 12 most of these mutants consist of substitutions at a small number of residues. In early studies, Frere and colleagues 13 showed that at least some mutant TEM enzymes had 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: b-shoichet@northwestern.edu Abbreviations used: WT, wild-type; ESBL, extended spectrum b-lactamase; IRT, inhibitor resistance TEM; RT, room temperature; FAP, 6-furylacrylpenicillanic acid; CAZ, ceftazidime; CTX, cefotaxime. † http://www.lahey.org/studies/webt.htm doi:10.1016/S0022-2836(02)00400-X available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 85–95