Assays for beta-lactamase activity and inhibition.

The ability, either innate or acquired, to produce beta-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in beta-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of beta-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn2+-dependent for their function.

68Zn isotope exchange experiments reveal an unusual kinetic lability of the metal ions in the di-zinc form of IMP-1 metallo-beta-lactamase.

The apparently paradoxical behaviour of facile exchange (kinetic lability) of tightly bound (thermodynamic stability) zinc ions in the enzyme IMP-1 metallo-beta-lactamase with Zn-68 and cadmium ions, as indicated by in-torch vaporization inductively-coupled plasma mass spectrometry (ITV-ICP-MS) and electrospray-ionization mass spectrometry (ESI-MS), is consistent with the involvement of a third metal ion in promoting Lewis acid/base type exchange processes.

Overexpression of isocitrate lyase is an important strategy in the survival of Pseudomonas fluorescens exposed to aluminum.

Isocitrate lyase, ICL (EC 4.1.3.

Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-beta-lactamases.

The inhibitory effect of a variety of thiol compounds on the function of binuclear metallo-beta-lactamases, with a particular focus on IMP-1 from Pseudomonas aeruginosa, has been investigated. Thiol inhibitors, depending on their structural features, fall into two categories, one in which inhibition at neutral pH was instantaneous and the other in which inhibition was time-dependent. While mercaptans with anionic substituents in the vicinity of their SH groups exhibited the former type of inhibition, neutral thiols appear to induce a slow, time-dependent isomerization of the initially formed EI complex to a tighter EI complex.

N-arylsulfonyl hydrazones as inhibitors of IMP-1 metallo-beta-lactamase.

Members of a family of N-arylsulfonyl hydrazones have been identified as novel inhibitors of IMP-1, a metallo-beta-lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efficient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo-beta-lactamase activity exhibited by this class of compounds.

IMP-1 metallo-beta-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry.

Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme.

Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence.

Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function.