Reactivity and Reaction Order in Acylhomoserine Lactone Formation by Pseudomonas aeruginosa RhlI

Abstract

The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.0 +/- 0.2 s (-1) and decayed with a rate constant of 3.7 +/- 0.2 s (-1). No other intermediates were detected, demonstrating that the RhlI reaction proceeds via acylation of S-adenosylmethionine, followed by lactonization. S-Adenosylhomocysteine acted as a pseudosubstrate, in that it did not undergo either acylation or lactonization but did induce the deacylation of butyryl-acyl carrier protein. The K m for S-adenosylhomocysteine was approximately 15-fold higher than the K m for S-adenosylmethionine. The reactivities of acylated and unacylated sulfonium ions that were analogues of S-adenosylmethionine were investigated by computational methods. The calculations indicated that acylation of the substrate amino group activated the substrate for lactonization, by allowing the carboxyl group oxygen to approach more closely the methylene carbon to which it adds. This observation provides a satisfying chemical rationale for the order of the individual reactions in the catalytic cycle.

A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases

“…These enzymes carry out unique reactions (4,5,8,9). We have been interested in identifying acyl-HSL synthase inhibitors to use as chemical probes for understanding the mechanism of enzyme activity, as tools to manipulate quorum sensing in the laboratory setting, and as potential scaffolds for therapeutic development.…”

“…Most of our knowledge about the enzymology of acyl-HSL synthesis is from studies of RhlI from Pseudomonas aeruginosa. Product-inhibition kinetics show that RhlI catalysis follows a sequential ordered bi ter mechanism (4,5). The transfer reaction occurs before lactonization with an acyl-SAM intermediate (5) and yields threeproducts: acyl-HSL, 5′-methylthioadenosine (MTA), and holo-ACP.…”

“…Product-inhibition kinetics show that RhlI catalysis follows a sequential ordered bi ter mechanism (4,5). The transfer reaction occurs before lactonization with an acyl-SAM intermediate (5) and yields threeproducts: acyl-HSL, 5′-methylthioadenosine (MTA), and holo-ACP. LuxItype synthases are considered as members of the Gcn5-related N-acetyltransferase superfamily (6), and yet they catalyze a unique reaction unlike other characterized enzymes in this superfamily (7).…”

“…A radiotracer has been developed, but this also requires sample manipulation and with respect to screening there are safety and regulatory issues. An assay in which the product holo-ACP is monitored with a thiol reagent has been developed, but it is not well suited to screening efforts because of limited sensitivity and interference with the absorbance readout by test compounds (4,5,8,9).…”

Frontier studies on highly selective bio-regulators useful for environmentally benign agricultural production

“…5, this catalytic reaction is thought to be proceeded by a twostep mechanism involving the intermediate acyl-ACP to the amino group of SAM and lactonization of the methionine part, concomitant with the release of methylthioadenosine (MTA). 71) We speculated that acyl-SAM analogs may be good candidates for competitive inhibitors of AHL synthases because many examples of catalytic intermediate analogs were previously reported to be good inhibitors of target enzymes, 72) and the intermediate acyl-SAM was shown to be specific to the synthesis of AHL in Gramnegative bacteria. 71) Furthermore, acyl-SAM has not yet been detected in other organisms; thus, these analogs may also be useful in crop protection from plant pathogens.…”