Journal of Molecular Biology
A Structure-based Model of the Reaction Catalyzed by Lumazine Synthase from Aquifex aeolicus
Introduction
The penultimate step of the riboflavin biosynthesis in the hyperthermophilic bacterium Aquifex aeolicus is catalyzed by lumazine synthase (LS), which is an assembly of 60 subunits arranged in a capsid with T=1 icosahedral symmetry. In the wild-type Bacillus subtilis enzyme, 60 LS subunits enclose a trimer of riboflavin synthase (RS), which catalyzes the dismutation of 6,7-dimethyl-8-ribityllumazine (3) resulting in the formation of riboflavin (4) and 5-amino-6-ribityl-amino-2,4(1H,3H)pyrimidinedione (1),1 (see Figure 1).
The three-dimensional structures of several lumazine synthases from bacteria, yeast and plants have been studied by X-ray crystallography.2., 3., 4., 5., 6., 7., 8. However, the precise mechanism of the catalytic reaction has remained unclear. Most of the existing functional and kinetic data have been obtained from studies on lumazine synthase from B. subtilis.9 Because of high structural homology (48% sequence identity), these results can be considered as valid for A. aeolicus LS (LSAQ), too, and will be reviewed in the following section.
The active site, which is located at the interface of two adjacent subunits in close proximity to the inner surface of the icosahedral capsid, exists in 60 copies with the same environment.2., 7. The enzymatic function of the LS had remained unknown for several decades. The chemical nature of the four-carbon precursor of the pyrazine ring of 6,7-dimethyl-8-ribityl-lumazine (3) was not understood until Volk & Bacher10., 11. identified this compound as (3S)-3,4-dihydroxy-2-butanone-4-phosphate (2; Figure 1), and established the enzyme-catalyzed formation of 6,7-dimethyl-8-ribityl-lumazine (3) from the butanone-phosphate (2) and the pyrimidinedione (1).
Kinetic and spectroscopic studies by Kis et al.12 have revealed a number of mechanistic details of the reaction catalyzed by LS. It has been shown that both enantiomers of the butanone-phosphate (2) can serve as substrates for LS; however, the reaction rate of the natural S-enantiomer was about sixfold higher than that for the R-enantiomer. The Km value for substrate 2 (130 μM) exceeded the Km value for the pyrimidine substrate 1 (5 μM) by more than an order of magnitude, which suggested an ordered bi–bi kinetic mechanism, but conclusive evidence has not been given. Strict regio-specificity was detected for the enzyme-catalyzed condensation of the carbohydrate-phosphate with the pyrimidinedione. The methyl protons of substrate 2 exchange spontaneously with the solvent as shown by NMR analysis.13., 14. In the absence of the pyrimidine substrate 1 the enzyme did not react with the carbohydrate substrate 2. More specifically, the enzyme complex did not catalyze the exchange of the proton at C-3 of substrate 2 with solvent water, nor did it act as a racemase. It was thus concluded that the initial step of the enzyme-catalyzed reaction requires the presence of the pyrimidine substrate 1 in the active site. A reaction mechanism initiated by the formation of a Schiff base upon the reaction of the 5-amino group of the pyrimidine substrate 1 with the carbonyl group of substrate 2 has been proposed. It has been assumed that this step would be followed by proton abstraction and phosphate elimination with the resulting double bond, thus being in favorable conjugation with the pyrimidine system. The enolate intermediate could then tautomerize under formation of a carbonyl group, which then could be attacked by the 6-amino group, resulting in ring closure. Finally, the release of water would terminate the reaction, resulting in energetically favorable conjugation in the heterocyclic double ring system of product 3.
The structures of the LS active site and the binding sites of substrates 1 and 2 have been analyzed earlier in complexes of LS from B. subtilis, Saccharomyces cerevisiae, Spinacea oleracea and Magnaporthe grisea with the substrate-analogs 5-nitroso-6-ribityl-amino-2,4(1H,3H)pyrimidinedione (RNOP; Figure 2(c)),2 5-nitro-6-ribityl-amino-2,4(1H,3H)pyrimidinedione (RNO2P; Figure 2(c))3., 4., 15. and 5-(6-d-ribitylamino-2,4(1H,3H)pyrimidinedione-5-yl)-1-pentyl-phosphonic acid (RPP; Figure 2(d)),6 respectively. These studies indicated that the side-chain of highly conserved Arg127 is involved in binding of the phosphate group of substrate 2. The pyrimidine ring of substrate 1 was found in offset stacking aromatic interaction with the ring system of a phenylalanine or alternatively a tryptophane indole ring system (Phe22/Trp22). Several hydrogen bond interactions to the pyrimidine system and the ribityl chain hydroxyl groups of substrate 1 are formed upon binding. They have been analyzed in S. cerevisiae LS and described in detail.6 The structure of an empty LS active site has been studied and described in LS from Brucella abortus5 and A. aeolicus.7 The observed conformational changes, obtained from comparisons with the occupied active sites, have suggested an induced-fit mechanism of substrate binding in LS. Several side-chains undergo specific conformational changes upon binding of the pyrimidine substrate; among the residues the highly conserved His88 (93% among 59 amino acid sequences; for an amino acid sequence alignment see Supplementary Material) suggests a critical involvement in catalysis. The aromatic ring of Phe22 adapts to the binding of the pyrimidine system by a swing movement, which results in a stacking aromatic interaction and proper orientation of the pyrimidine.
In this work we have determined and refined at high resolution the crystal structures of four complexes of A. aeolicus LS with substrate and product analogous inhibitors, among them for the first time an analogue of the product 6,7-dimethyl-8-ribityl-lumazine (3). We analyze and describe in detail the binding modes of these inhibitors. By taking into account the constraints imposed by the active site structure, the functional data from single-site mutants of the highly homologous B. subtilis LS16 and the conservation of distinct amino acid side-chains in the active site, a structural model of the catalytic process, which illustrates binding of both substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization will be proposed.
Section snippets
Overall structure and quality of the refined models
The structures of icosahedral lumazine synthase from A. aeolicus (LSAQ) in complex with the inhibitors 6,7-dioxo-5H-8-ribitylaminolumazine (RDL; Figure 2(a)), 3-(7-hydroxy-8-ribityllumazine-6-yl)propionic acid (RPL; Figure 2(b)), 6-ribitylamino-5-nitroso-2,4(1H,3H)pyrimidindione (RNOP; Figure 2(c)), and 5-(6-d-ribitylamino-2,4(1H,3H) pyrimidinedione-5-yl)pentyl-1-phosphonic acid (RPP; Figure 2(d)) were solved by molecular replacement using the native structure of LSAQ7 as a search model. The
Materials
RDL was prepared as described;23 RPL was synthesized by Cushman et al. (M.C., unpublished results); RNOP was synthesized with published procedures;24 RPP was prepared as described by Cushman et al.25
Molecular biological and enzymological methods
LS from A. aeolicus was prepared as described.7 The reaction mixtures were incubated at 50 °C. The protein concentration was determined by measuring the absorbance at 280 nm
Crystallization, co-crystallization and crystal soaking
The purified protein was concentrated to 15 mg/ml and stored in a solution containing 0.5 mM EDTA, 0.5 mM
Supplementary Files
Acknowledgements
This work was supported by grants from the Swedish Natural Science Research Council, NIH grant GM1469, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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- †
X.Z. & W.M. contributed equally to this work.