Catalytic mechanism of S-ribosylhomocysteinase (LuxS): direct observation of ketone intermediates by 13C NMR spectroscopy.
S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce l-homocysteine and 4,5-dihydroxy-2,3-pentanedione (DHPD). This is a key step in the biosynthetic pathway of the type II autoinducer (AI-2) in both Gram-positive and Gram-negative bacteria. Previous studies demonstrated that LuxS contains a catalytically essential Fe2+ ion. The catalytic mechanism of LuxS was investigated using 2- and 3-13C-labeled SRH as substrate and 13C NMR spectroscopy. These studies revealed the presence of 2- and 3-keto intermediates in the catalytic pathway. The 2-keto intermediate was chemically synthesized, and its chemical and kinetic competence was demonstrated. The results support a catalytic mechanism in which the metal ion catalyzes an internal redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position. Subsequent beta-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol. The results also suggest that Cys-84 and Glu-57 are the possible general acids/bases for proton transfer during catalysis and that the keto intermediates are released from the enzyme active site before rebinding and completion of the reaction.
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Jinge
J
Zhu
Xubo
X
Hu
Eric
E
Dizin
Dehua
D
Pei
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D013437:Carbon-Sulfur Lyases; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007659:Ketones / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D007659:Ketones; D003035:Cobalt; D006867:Hydrolases; C463961:homocysteinase; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria