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.
Zhu
Jinge
J
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Hu
Xubo
X
Dizin
Eric
E
Pei
Dehua
D
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