A continuous kinetic assay for adenylation enzyme activity and inhibition

Abstract

Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PP i ). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatasepurine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate-MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/ or fail to undergo rapid ATP-PP i exchange.

Baulamycins A and B, Broad-Spectrum Antibiotics Identified as Inhibitors of Siderophore Biosynthesis in Staphylococcus aureus and Bacillus anthracis

“…43,44 These experiments were conducted by varying a single substrate and holding remaining reaction components at a constant saturating level in the presence and absence of inhibitor. The resulting double reciprocal (Lineweaver-Burk) plots confirmed that kinetic parameters closely matched previously reported values for both enzymes against varied citrate concentrations (Table S1).…”

Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis

“…To accomplish this, ATP turnover from synthetase activity was monitored using an enzyme-coupled assay involving conversion of the reporter molecule MESG to its purine base (44,45). This assay was used to empirically establish saturating levels of spermidine, ATP, and Mg 2ϩ for AsbA to determine reaction rates dependent exclusively on citric acid.…”

“…2-Amino-6-mercapto-7-methylpurine Ribonucleoside (MESG) Continuous Pyrophosphate Detection Assay-Reactions were a modification of a previously described protocol (44,45). For kinetics reactions, standard conditions were 50 mM Tris, pH 8.0, 15 mM MgCl 2 , 0.5 mM tris(2-carboxyethyl) phosphine, 6 mM ATP, 40 mM spermidine, 0.001 units/l purine nucleoside phosphorylase, 0.0004 units/l inorganic pyrophosphatase, 0.4 mM MESG (Berry and Associates, Dexter, MI), and 0.2 M purified enzyme.…”

Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases

“…TycB1 is derived from the tyrocidine biosynthetic enzymes, incorporates l -Pro, and contains the domain structure C-A ( l -Pro)-T. 18 Apparent K i values were determined by a coupled hydroxamate-MesG continuous spectrophotometric assay (Figure S1). 19 Probes 1 and 2 exhibited tight-binding properties against the A domains of GrsA and TycB 1 , respectively, with calculated K i app values of 1.43 ± 0.19 nM for GrsA and 327 ± 17.0 nM for TycB1 (Figure S2). Inhibitors 4 and 5 gave K i app values of 1.20 ± 0.14 nM for GrsA 16 and 431 ± 42.0 nM for TycB1, respectively (Figure S2).…”