Mechanistic and inhibitory studies of S-ribosylhomocysteinase (LuxS)

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine to produce L-homocysteine and 4,5-dihydroxy-2,3-pentanedione, the precursor of type II bacterial quorum sensing autoinducer. No mechanistic hypothesis of the LuxS-catalyzed reaction was available prior to our work. In this work, substantial mechanistic studies of the LuxS reaction were carried out and specific inhibitors against LuxS were designed and tested.

The native metal cofactor of LuxS was identified as Fe²⁺, instead of previously reported Zn²⁺, and oxidation of Fe²⁺ was proved to be responsible for enzyme instability and cysteine-84 oxidation. Spectroscopic studies of Co²⁺-LuxS suggested a catalytic role for the metal, and the co-crystal structure of C84A Co²⁺-LuxS from Bacillus subtilis in complex with a 2-ketone intermediate provided strong structural support for the Lewis acid function of the metal ion.

Based on the X-ray crystal structures and preliminary studies, a catalytic mechanism of the LuxS reaction was proposed, which involved an internal redox reaction via two consecutive carbonyl migration steps followed by β-elimination, and was supported by following studies. First, the 2-keto and 3-keto intermediates were directly observed by real time 13C NMR spectroscopy and our data suggest that the keto intermediates are at least partially released from the active site during catalysis. In addition, 2-ketone was chemically synthesized and demonstrated to be chemically and kinetically competent on the LuxS catalytic pathway. Second, reaction in D₂O resulted in the incorporation of deuterium into product, supporting the proton transfer events. Furthermore, the regiochemical and stereochemical course and the primary kinetic isotope effect of the proton transfer reactions were examined with individually deuterium-labeled substrates. Finally, mutagenesis studies established Cys-84 and Glu-57 as critical residues for catalysis, likely acting as the first and second general acids/bases, respectively. The pKa values of Cys-84 and metal-bound H₂O were determined by absorption spectroscopy.

Three LuxS activity assays were developed and greatly facilitated the mechanistic investigations of LuxS. Two classes of LuxS inhibitors were designed based on metal chelation and catalytic mechanism, respectively. They encouraged future development of LuxS inhibitors as novel antibacterial agents and helped probe the catalytic mechanism of LuxS.