The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV β-lactamase: A study of Ambler position Asn276

Inhibitor-resistant class A β-lactamases of the TEM and SHV families that arise by single aminoacid substitutions are a significant threat to the efficacy of β-lactam/β-lactamase inhibitor combinations. Tobetter understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis toexamine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only theAsn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2f 50/8 μg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 μg/mL). Steady-statekinetic analyses of Asn276Asp revealed slightly diminished kcat/Km for all substrates tested. In contrast, weobserved a 5-fold increase in Ki for clavulanate (7.4(0.9 μMfor Asn276Asp vs 1.4(0.2 μMfor SHV-1) and a40% reduction in kinact/KI (0.013 ( 0.002 μM-1 s-1 for Asn276Asp vs 0.021 ( 0.004 μM-1 s-1 for SHV-1).Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Aspshowed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modelingshows that novel electrostatic interactions are formed between Arg244Nη2 and both 276AspOδ1 and Oδ2;these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C3/C4carboxylate of β-lactam substrates and inhibitors. Testing the functional consequences of this interaction, wenoted considerable free energy costs (+ΔΔG) for substrates and inhibitors. A rigid carbapenem (meropenem)was most affected by the Asn276Asp substitution (46-fold increase in Ki vs SHV-1). We conclude that residue276 is an important second-shell residue in class A β-lactamase-mediated resistance to substrates andinhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required forsuccessful evolution in nature.