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Lumazine synthase

Lumazine synthase (LS) catalyzes the penultimate step in the biosynthesis of riboflavin. Coenzymes derived from riboflavin are indispensable in all cellular organisms. ¹ Enterobacteriaceae and yeasts are also absolutely dependent on the endogenous synthesis of riboflavin because they are devoid of an efficient uptake system. ² These findings make lumazine synthase an attractive drug target.

Lumazine synthase can assume various quaternary structures. Among the assembly forms, which are structurally characterized, so far, are hollow capsids with icosahedral symmetry constituted by 60 identical subunits, ^3,4,5,6 pentamers, ^6,7,8,9 and dimers of two tightly packed pentamers. ^10,11 Besides these forms there is indication for at least two more larger capsids forms. The exact stoichiometry of these forms has so far remained unclear. The B. subtilis lumazine synthase/riboflavin synthase complex reassembles under certain conditions to large hollow capsids with a diameter of about 330 Å. Empty T=1 capsids can be reconstituted in presence of a substrate analogue that binds to the active site. ¹² The subunit fold and the arrangement of subunits in pentameric building blocks are highly conserved among all known LS structures.

Structural studies aiding in the elucidation of the reaction mechanism of lumazine synthase and the development of novel drug candidates

We have solved the structures of lumazine synthases from Saccharomyces cerevisiae ⁷ and Aquifex aeolicus ^3,13 both in complex with different substrate and intermediate analogs and in their native form. The structure of the A. aeolicus LS is the first structure of a lumazine synthase from a hyperthermophilic organism. We performed an in dept study of 4 different complexes of A. aeolicus lumazine synthase with substrate and intermediate analogs. ¹³ This study lead to the first structure based suggestion of how the conversion catalyzed by lumazine synthase might proceed.

Stability and thermodynamics of lumazine synthases

In order to study structural differences and to relate them to differences in thermodynamic stability of lumazine synthases we analyzed ion pairs and ion pair networks and the properties of accessible surfaces in a comparative study of different lumazine synthases. ³ The native structure of lumazine synthase from A. aeolicus was determined and compared to the lumazine synthases from B. subtilis, Sp. oleracea, S. cerevisiae, B. abortus, and M. grisea. The increased number of ion pair networks as well as the accessible surface formed by charged residues clearly correlated with the increase of the melting temperature of lumazine synthases from the mesophilic to the hyperthermophilic species. This finding is well in line with the earlier observation that the optimization of hydrophobic and ionic contacts plays a role in gaining thermostability.

Multiple assembly states of lumazine synthase

In a recent study we found that several native and modified lumazine synthases can form larger capsid-like assemblies. ¹⁴ The native enzymes from both B. subtilis and A. aeolicus appear when analyzed by small angle scattering and electron microscopy as mixtures of smaller and larger capsids, which are partially deformed or incomplete. The relative abundance of these forms strongly depends on buffer and pH of the solution. The smaller capsid form of the B. subtilis is stabilized in presence of inorganic phosphate, whereas in other buffers the same enzyme assembles to larger capsids. Native A. aeolicus LS appears to be a mixture of both small and larger capsids independently of pH or buffer. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 Å. These mutants also reduce or inhibit enzymatic activity suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure.

Structure of a mutant of A. aeolicus LS with a four residue insertion

LSAQ-IDEA is a mutant form of LS from A. aeolicus. In this mutant a sequence of four residues Ile, Asp, Glu and Ala is inserted after residue 129. This insert matches the sequence of LS from S. cerevisiae in this sequence region. Nilsson et al. recently determined a low resolution reconstruction of LSAQ-IDEA and found that the capsid consists of 180 subunits following icosahedral symmetry. ¹⁵ The reconstruction indicates that the subunit contacts do not obey the principles of quasi-equivalent contacts as introduced by Caspar and Klug. ¹⁶ Several contact interfaces are different, which according to this theory should be similar. The comparison shows that the inner channel of the pentameric substructures in the T=3 capsid is expanded with respect to the corresponding channel of the native enzyme. The channel diameter increases from 9Å to 19 Å and the diameter of the pentamer block increases by 8 Å from 72 Å to 80 Å.

References

1.          Bacher, A. (1991). In Chem. Biochem. Flavoenzymes (Müller, F., ed.), Vol. I, pp. 215-259. Chemical Rubber & Co., Boca Raton, Florida.

2.          Kearny, E. B., Goldenberg, J., Lipsick, J. & Perl, M. (1979). J. Biol. Chem. 254, 9551-9557.

3.          Zhang, X., Meining, W., Fischer, M., Bacher, A. & Ladenstein, R. (2001). J. Mol. Biol. 306, 1099-114.

4.          Ladenstein, R., Schneider, M., Huber, R., Bartunik, H. D., Wilson, K., Schott, K. & Bacher, A. (1988). J. Mol. Biol. 203, 1045-1070.

5.          Mörtl, S., Fischer, M., Richter, G., Tack, J., Weinkauf, S. & Bacher, A. (1996). J. Biol. Chem. 271, 33201-33207.

6.          Persson, K., Schneider, G., Jordan, D. B., Viitanen, P. V. & Sandalova, T. (1999). Protein Sci. 8, 2355-2365.

7.          Meining, W., Mörtl, S., Fischer, M., Cushman, M., Bacher, A. & Ladenstein, R. (2000). J. Mol. Biol. 299, 181-97.

8.          Gerhardt, S., Haase, I., Steinbacher, S., Kaiser, J. T., Cushman, M., Bacher, A., Huber, R. & Fischer, M. (2002). J. Mol. Biol. 318, 1317-1329.

9.          Morgunova, E., Meining, W., Illarionov, B., Haase, I., Jin, G., Bacher, A., Cushman, M., Fischer, M. & Ladenstein, R. (2005). Biochemistry 44, 2746-58.

10.        Braden, B. C., Velikovsky, C. A., Cauerhff, A. A., Polikarpov, I. & Goldbaum, F. A. (2000). J. Mol. Biol. 297, 1031-1036.

11.        Zylberman, V., Craig, P. O., Klinke, S., Braden, B. C., Cauerhff, A. & Goldbaum, F. A. (2004). J. Biol. Chem. 279, 8093-101.

12.        Bacher, A., Ludwig, H. C., Schnepple, H. & Ben-Shaul, Y. (1986). J. Mol. Biol. 187, 75-86.

13.        Zhang, X., Meining, W., Cushman, M., Haase, I., Fischer, M., Bacher, A. & Ladenstein, R. (2003). J. Mol. Biol. 328, 167-82.

14.        Zhang, X., Konarev, P., Svergun, D., Haase, I., Fischer, M., Bacher, A., Ladenstein, R. & Meining, W. (2006). J. Mol. Biol. 362, 753-770.

15.        Nilsson, J., Xing, L., Zhang, X., Bergman, L., Haase, I., Fischer, M., Bacher, A., Meining, W., Ladenstein, R. & Cheng, R. H. (2006). manuscript in preparation.

16.        Caspar, D. L. D. & Klug, A. (1962). Cold Spring Harbor Symp. Quant. Biol. 27, 1-24.