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

Biochemistry. 2009 Jun 2;48(21):4557-66. doi: 10.1021/bi9003292.


Inhibitor-resistant class A beta-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of beta-lactam/beta-lactamase inhibitor combinations. To better understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis to examine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only the Asn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2 --> 50/8 microg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 microg/mL). Steady-state kinetic analyses of Asn276Asp revealed slightly diminished k(cat)/K(m) for all substrates tested. In contrast, we observed a 5-fold increase in K(i) for clavulanate (7.4 +/- 0.9 microM for Asn276Asp vs 1.4 +/- 0.2 microM for SHV-1) and a 40% reduction in k(inact)/K(I) (0.013 +/- 0.002 microM(-1 )s(-1) for Asn276Asp vs 0.021 +/- 0.004 microM(-1) s(-1) for SHV-1). Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Asp showed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modeling shows that novel electrostatic interactions are formed between Arg244Neta2 and both 276AspOdelta1 and Odelta2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C(3)/C(4) carboxylate of beta-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs (+DeltaDeltaG) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in K(i) vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A beta-lactamase-mediated resistance to substrates and inhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required for successful evolution in nature.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anti-Infective Agents / metabolism
  • Anti-Infective Agents / pharmacology
  • Asparagine*
  • Catalytic Domain
  • Clavulanic Acid / metabolism
  • Clavulanic Acid / pharmacology
  • Enzyme Inhibitors / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / drug effects
  • Humans
  • Immunoblotting
  • Kinetics
  • Mutagenesis
  • Protein Binding
  • Spectrometry, Mass, Electrospray Ionization
  • beta-Lactamase Inhibitors*
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism*


  • Anti-Infective Agents
  • Enzyme Inhibitors
  • beta-Lactamase Inhibitors
  • Clavulanic Acid
  • Asparagine
  • beta-Lactamases