title: Probing the catalytic mechanism of S-ribosylhomocysteinase (LuxS) with catalytic intermediates and substrate analogues.
abstract: S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.
authors.affiliation: Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Gopishetty
authors.forename: Jinge
authors.initials: J
authors.lastname: Zhu
authors.forename: Rakhi
authors.initials: R
authors.lastname: Rajan
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Sobczak
authors.forename: Stanislaw F
authors.initials: SF
authors.lastname: Wnuk
authors.forename: Charles E
authors.initials: CE
authors.lastname: Bell
authors.forename: Dehua
authors.initials: D
authors.lastname: Pei
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism*; D055162:Biocatalysis; D013437:Carbon-Sulfur Lyases / Q000032:analysis* / Q000378:metabolism*; D020134:Catalytic Domain; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D007659:Ketones; D006710:Homocysteine; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria