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.
Drawz
Sarah M
SM
Department of Pathology, CaseWestern Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Bethel
Christopher R
CR
Hujer
Kristine M
KM
Hurless
Kelly N
KN
Distler
Anne M
AM
Caselli
Emilia
E
Prati
Fabio
F
Bonomo
Robert A
RA
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