Crystal Structure of S-Ribosylhomocysteinase (LuxS) in Complex with a Catalytic 2-Ketone Intermediate^,

Abstract

S-Ribosylhomocysteinase (LuxS) is an Fe(2+)-dependent metalloenzyme that catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum-sensing molecule. The proposed mechanism involves an initial metal-catalyzed aldose-ketose isomerization reaction, which results in the migration of the ribose carbonyl group from its C1 to C2 position and the formation of a 2-ketone intermediate. A repetition of the isomerization reaction shifts the carbonyl group to the C3 position. Subsequent beta-elimination reaction at the C4 and C5 positions completes the catalytic cycle. In this work, a catalytically inactive mutant (C84A) of Co(2+)-substituted Bacillus subtilis LuxS was cocrystallized with the 2-ketone intermediate and the structure was determined to 1.8 A resolution. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis. Cys-84 and Glu-57 are optimally positioned to act as general acids/bases during the isomerization and elimination reactions. In addition, Ser-6, His-11, and Arg-39 are involved in substrate/ intermediate binding through hydrogen bonding interactions. The above conclusions are further confirmed by site-directed mutagenesis and visible absorption spectroscopic studies.

The 4-aza-S-ribosyl-L-homocysteine Derivatives and the Related Gamma-lactam and Azahemiacetal Analogs: Synthesis, Inhibition and Quorum Sensing Activity

“…The function of the metal as a Lewis acid was established from the crystal structure of LuxS with the 2-keto intermediate, 3 (Scheme 3). 49 The proposed metal mediated catalysis was confirmed by the dramatic change in electronic absorption spectrum of cobalt ion when the natural substrate, SRH was added to the Co-LuxS Bs (from peaks at 530, 570, 650 nm to none). 50 Interestingly, Cys84 which is highly conserved in the active site, was found oxidized to cysteine sulfonic acid and cysteine sulfinic acids leading to rapid inactivation of LuxS, suggesting complexation of metal ion to Cys84 in catalysis.…”

“…Crystal structure of LuxS Bs in complex with 2-keto intermediate. 49 The crystal structure of LuxS Bs in complex with SRH at 2.2 A° and homocysteine at 2.3 A° respectively, was determined by Baker et al 46 "The homocysteine moiety of SRH is bound in an extended conformation with the amino and carboxyl groups stabilized by hydrogen bonds with five neighboring residues from helix α1 and strand β3 (Arg65, Asp78, Ile79, Ser80 and Gly127 and Lys35 of neighboring subunit). The ribose moiety appears to be in the C2ˈ-endo conformation and the ribose O2 and O3 hydroxyl groups can be seen involved in long ion-dipole interactions with the Zn 2+ ion.…”

“…94,95 Cyclic ketones, 48a, 48b were found to reduce immunomodulatory responses (for e.g., TNFα factor and nitric oxide production) ( Figure 10). Muh et al screened a library of 200,000 compounds using ultra-high-throughput-cell based screening and identified two LasR inhibitors having a phenyl (49) and tetrazole ring (50) connected to 12 carbon aliphatic chain similar to 3-oxo-C 12 -AHL ( Figure 11). 96 Phenyl analog 49…”

“…This hydroxylic group was originally shown to interact with several active site residues (Ser-6, His-11 and Arg-39) via hydrogen bonding in the co-crystal structure of C84A-LuxS Bs with 2-ketone intermediate. 49 Because of the positively charged ring nitrogen (~pK a = 6.6), the conformational change in sugar ring might have also resulted in improper fit into the active site. form new interactions at the active site via hydrogen bonding.…”

“…In the third step, base/acid abstracts proton at C4 with elimination of homocysteine to give an enol intermediate which ultimately tautomerizes to DPD. 49,50,53,54 The…”