Over-expression, purification, and characterization of metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Crawford, Patrick A.; Sharma, Narayan P.; Chandrasekar, Sowmya; Sigdel, Tara K.; Walsh, Timothy R.; Spencer, James; Crowder, Michael W.

doi:10.1016/j.pep.2004.04.017

Cited by 50 publications

(53 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous steady-state kinetic studies have shown that the recombinant ImiS utilized for the present studies displays catalytic constants similar to those found for native ImiS (78) and CphA (73) and that ImiS shows a preference for carbapenem substrates, typical of class B2 M Ls. The first equivalent of Zn binds to ImiS with a K d < 2 nM (78).…”

Section: Resultssupporting

confidence: 75%

“…One-and two-Zn forms of wild-type ImiS were prepared and characterized kinetically according to published procedures (78). Using the PAGE-purified oligonucleotide primers and the overexpression plasmid for ImiS (pET26b-imiS) as a template, the ImiS mutant was generated using the Quikchange site-directed mutagenesis kit, according to the instructions of the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

“…To test for the overexpression of mutants, the mutant plasmids were transformed into Escherichia coli BL21(DE3) cells and small-scale cell cultures were grown. Metal content and steady-state kinetic constants were determined as previously reported (78).…”

Section: Methodsmentioning

confidence: 99%

“…The B2 enzymes are less well-characterized, with comparably fewer physicochemical studies available (73)(74)(75)(76)(77)(78)(79)(80)(81). Recently, the first crystal structure of a B2 enzyme was reported (82).…”

mentioning

confidence: 99%

“…An alternative model, suggesting that the catalytic metal ion is either labile (distributed between the two sides of the active site) or resides in a site made up of ligands from both sites has been put forth for AE036 from A. hydrophila (77). Kinetic data for both CphA and ImiS show that a second equivalent of zinc acts as a noncompetitive inhibitor of enzymatic activity (76,78). At present, the only structural information regarding this second, inhibitory binding site is for the cobalt analogue of ImiS (79), where electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) data were interpreted as indicative of a remote (>7 Å) binding site for the inhibitory metal ion.…”

mentioning

confidence: 99%

See 4 more Smart Citations

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

et al. 2006

View full text Add to dashboard Cite

X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas Veronii bV. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn 2 site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345,[785][786][787][788][789][790][791][792][793][794][795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct R3 helix, and H118, a canonical Zn 1 ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.

show abstract

Section: Resultssupporting

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

et al. 2006

View full text Add to dashboard Cite

show abstract

Metallo‐β‐lactamases and their Synthetic Mimics: Structure, Function, and Catalytic Mechanism

Umayal

Tamilselvi

Mugesh

2011

Progress in Inorganic Chemistry

View full text Add to dashboard Cite

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

et al. 2006

View full text Add to dashboard Cite

Extended X-ray absorption fine structure studies of the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn1 site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal-metal interaction at 3.42 A. Reaction with the beta-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn-Zn interaction to 3.62 A.

show abstract

Over-expression, purification, and characterization of metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Cited by 50 publications

References 55 publications

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Metallo‐β‐lactamases and their Synthetic Mimics: Structure, Function, and Catalytic Mechanism

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Contact Info

Product

Resources

About