S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein.
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 Zn(2+) 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 Fe(2+), Co(2+), or Zn(2+). 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 Fe(2+). 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 D(2)O 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 beta-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol.
Zhu
Jinge
J
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Dizin
Eric
E
Hu
Xubo
X
Wavreille
Anne-Sophie
AS
Park
Junguk
J
Pei
Dehua
D
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases; D002384:Catalysis; D003903:Deuterium; D004795:Enzyme Stability; D006714:Homoserine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007783:Lactones; D008670:Metals / Q000032:analysis; D008956:Models, Chemical; D009154:Mutation
D001426:Bacterial Proteins; D033862:Iron-Binding Proteins; D007783:Lactones; D008670:Metals; C091088:N-octanoylhomoserine lactone; D006714:Homoserine; D003903:Deuterium; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria