The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV beta-lactamase: a study of ambler position Asn276.
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.
Department of Pathology, CaseWestern Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Sarah M
SM
Drawz
Christopher R
CR
Bethel
Kristine M
KM
Hujer
Kelly N
KN
Hurless
Anne M
AM
Distler
Emilia
E
Caselli
Fabio
F
Prati
Robert A
RA
Bonomo
D000890:Anti-Infective Agents / Q000378:metabolism / Q000494:pharmacology; D001216:Asparagine; D020134:Catalytic Domain; D019818:Clavulanic Acid / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D016296:Mutagenesis; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D001216:Asparagine; D001618:beta-Lactamases