Kinetic Comparison of the Specificity of the Vancomycin Resistance Kinase VanS for Two Response Regulators, VanR and PhoB

Abstract

Induction of vancomycin resistance in the Gram-positive Enterococci requires a two-componet regulatory system, VanS and VanR, for transcriptional activation of three genes (vanH, A, X) that encode enzymes for a cell wall biosynthetic pathway that produces an altered peptidoglycan intermediate with lower affinity for the antibiotic. The catalytic efficiency (kcat/KM) has been determined for phosphotransfer from the phosphohistidyl form of VanS to both its homologous partner VanR and the heterologous (Escherichia coli) response regulator Phob. The rate of formation of the phosphoaapartyl forms of VanR and PhoB were determined as well as the rate of appearance of inorganic phosphate. Using PhoB in excess of P-VanS, a pseudo-first-order rate constant (kxfer) of 0.2 min-1 for phosphotransfer and a KM for PhoB of 100 microM were readily determined. The corresponding kxfer of 96 min-1 for phosphotransfer from P-VanS to VanR required quench kinetics. A KM of 3 microM was estimated for VanR, leading to a 10(4)-fold preference in kxfer/KM for phosphotransfer to VanR compared to PhoB. No phosphotransfer was detachable to three other E. coli response regulators, OmpR, ArcB, or CreB, providing some sense of the selectivity against two-component regulatory system cross-talk. In the phosphotransfer from P-VanS to PhoB and VanR, there was evidence of competition between water, to give Pi, and the specific aspartyl beta-COO- moiety of either PhoB or VanR with about 25% of the initial flux generating inorganic phosphate. The kinetics of phosphotransfer from P-VanS to VanR were complicated by inhibition by free VanS but, the inhibition pattern could be modeled to yield at KD of 30 nM for VanR binding to free VanS, an affinity similar to that of the CheA-CheY pair in E. coli chemotaxis.

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB

“…For that reason, k dephos (het) was also set equal to the rate constant determined for [ 32 P]PhoB N R115D-P. Note that the measured k dephos values for PhoB N are in close agreement with previously reported values for k dephos for PhoB F (30,35,39).…”

Crosstalk and the evolution of specificity in two-component signaling

“…Bacterial TCS networks show a remarkably different topology: HKs usually act on a single target (12)(13)(14)(15)(16)(17). Intensive experimental study over the past 10 years has revealed the biochemical and biophysical basis for this lack of promiscuity.…”

In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli

“…A His 6 -tagged form of the CpxR protein, hereafter referred to simply as CpxR, was prepared as described previously (26). PhoB, a kind gift of M. Prahalad and C. T. Walsh (Harvard Medical School), was prepared as previously described (6).…”