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.

The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV β-lactamase: A study of Ambler position Asn276 / S. M., Drawz; C. R., Bethel; K. H., Hujer; K. N., Hurless; A. M., Distler; Caselli, Emilia; Prati, Fabio; R. A., Bonomo. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 48:21(2009), pp. 4557-4566. [10.1021/bi9003292]

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

CASELLI, Emilia;PRATI, Fabio;
2009

Abstract

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.
2009
48
21
4557
4566
The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV β-lactamase: A study of Ambler position Asn276 / S. M., Drawz; C. R., Bethel; K. H., Hujer; K. N., Hurless; A. M., Distler; Caselli, Emilia; Prati, Fabio; R. A., Bonomo. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 48:21(2009), pp. 4557-4566. [10.1021/bi9003292]
S. M., Drawz; C. R., Bethel; K. H., Hujer; K. N., Hurless; A. M., Distler; Caselli, Emilia; Prati, Fabio; R. A., Bonomo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/639768
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