A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1)

Pei W. Thomas, Michael Cammarata, Jennifer Brodbelt, Arthur F. Monzingo, R. F. Pratt, Walter Fast

Research output: Contribution to journalArticle

Abstract

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 μM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-β-lactamases.

Original languageEnglish (US)
Pages (from-to)2834-2843
Number of pages10
JournalBiochemistry
Volume58
Issue number25
DOIs
StatePublished - Jun 25 2019

Fingerprint

Affinity Labels
Serine
Lysine
Catalytic Domain
Enterobacter cloacae
Lactams
Site-Directed Mutagenesis
Photodissociation
Mutagenesis
Zinc
Mass Spectrometry
Proteins
Substrates
Binding Sites
X-Rays
Labeling
Dilution
Mass spectrometry
Assays
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1). / Thomas, Pei W.; Cammarata, Michael; Brodbelt, Jennifer; Monzingo, Arthur F.; Pratt, R. F.; Fast, Walter.

In: Biochemistry, Vol. 58, No. 25, 25.06.2019, p. 2834-2843.

Research output: Contribution to journalArticle

Thomas, Pei W. ; Cammarata, Michael ; Brodbelt, Jennifer ; Monzingo, Arthur F. ; Pratt, R. F. ; Fast, Walter. / A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1). In: Biochemistry. 2019 ; Vol. 58, No. 25. pp. 2834-2843.
@article{bbc1f834678440cd9fa7ee8d494fa13c,
title = "A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1)",
abstract = "The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 μM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 {\AA} resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-β-lactamases.",
author = "Thomas, {Pei W.} and Michael Cammarata and Jennifer Brodbelt and Monzingo, {Arthur F.} and Pratt, {R. F.} and Walter Fast",
year = "2019",
month = "6",
day = "25",
doi = "10.1021/acs.biochem.9b00393",
language = "English (US)",
volume = "58",
pages = "2834--2843",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "25",

}

TY - JOUR

T1 - A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1)

AU - Thomas, Pei W.

AU - Cammarata, Michael

AU - Brodbelt, Jennifer

AU - Monzingo, Arthur F.

AU - Pratt, R. F.

AU - Fast, Walter

PY - 2019/6/25

Y1 - 2019/6/25

N2 - The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 μM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-β-lactamases.

AB - The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-β-lactamases hamper the development of wide-spectrum β-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-β-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-β-lactamases have an analogous active-site Lys residue used to bind β-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-β-lactamases can also serve as a classical affinity label for New Delhi metallo-β-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 μM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-β-lactamases.

UR - http://www.scopus.com/inward/record.url?scp=85068191153&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85068191153&partnerID=8YFLogxK

U2 - 10.1021/acs.biochem.9b00393

DO - 10.1021/acs.biochem.9b00393

M3 - Article

C2 - 31145588

AN - SCOPUS:85068191153

VL - 58

SP - 2834

EP - 2843

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 25

ER -