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Structure and mechanism of copper- and nickel-substituted analogues of metallo-beta-lactamase L1. Biochemistry 2009 Apr 07;48(13):2981-9

Date

02/21/2009

Pubmed ID

19228020

Pubmed Central ID

PMC2671888

DOI

10.1021/bi802295z

Scopus ID

2-s2.0-65249135885 (requires institutional sign-in at Scopus site)   21 Citations

Abstract

In an effort to further probe metal binding to metallo-beta-lactamase L1 (mbetal L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin-spin coupling. Cu-L1 exhibited higher k(cat) (96 s(-1)) and K(m) (224 microM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equiv of Ni and 0.3 equiv of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by (1)H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. (1)H NMR spectra demonstrate that Ni(II) binds in the Zn(2) site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn(2) site modulates substrate preference in mbetal L1. These studies demonstrate that the Zn(2) site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with beta-lactam-containing antibiotics.

Author List

Hu Z, Spadafora LJ, Hajdin CE, Bennett B, Crowder MW

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Catalytic Domain
Copper
Electron Spin Resonance Spectroscopy
Kinetics
Magnetic Resonance Spectroscopy
Nickel
Protein Structure, Secondary
Spectrum Analysis
Stenotrophomonas maltophilia
Substrate Specificity
beta-Lactamases