S-Ribosylhomocysteinase (LuxS) Is a Mononuclear Iron Protein

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Abstract
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 divalent metal cofactor, which has been proposed to be a Zn2+ ion. To gain insight into the catalytic mechanism of this unusual reaction and the function of the metal cofactor, we developed an efficient expression and purification system to produce LuxS enriched in either Fe2+, Co2+, or Zn2+. Comparison of the catalytic properties and stability of the metal-substituted LuxS with those of the native enzyme revealed that the native metal ion is Fe2+. The electronic absorption spectrum of the Co(II)-substituted LuxS underwent dramatic catalysis-dependent changes, suggesting the direct involvement of the metal ion in catalysis. Site-directed mutagenesis studies showed that Glu-57 and Cys-84 are essential for catalysis, most likely acting as general acids/bases. Reaction in D2O resulted in the incorporation of deuterium at the C-1, C-2, and C-5 positions of the diketone product. These data suggest a catalytic mechanism in which the metal ion catalyzes an intramolecular redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position of the ribose. Subsequent β-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol.