| 1. |
- Meining, Winfried, et al.
(författare)
-
The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6 A resolution
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 331:5, s. 1053-1063
-
Tidskriftsartikel (refereegranskat)abstract
- Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here,we report the structure of this construct in complex with riboflavin at 2.6 Angstrom resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.
|
|
| 2. |
- Zhang, X F, et al.
(författare)
-
A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 328:1, s. 167-182
-
Tidskriftsartikel (refereegranskat)abstract
- 6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 Angstrom, 1.85 Angstrom, 2.05 Angstrom and 2.2 Angstrom, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of singlesite mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.
|
|
| 3. |
- Knapp, S, et al.
(författare)
-
Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus
- 1996
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 264:5, s. 1132-1144
-
Tidskriftsartikel (refereegranskat)abstract
- Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(mol K)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximate to 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximate to 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed. (C) 1996 Academic Press Limited
|
|
| 4. |
- Meining, Winfried, et al.
(författare)
-
The atomic structure of pentameric lumazine synthase from Saccharomyces cerevisiae at 1.85 angstrom resolution reveals the binding mode of a phosphonate intermediate analogue
- 2000
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 299:1, s. 181-197
-
Tidskriftsartikel (refereegranskat)abstract
- Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa. Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained. The space group is P4(1)2(1)2 with lattice dimensions 82.9 Angstrom * 82.9 Angstrom * 300.2 Angstrom. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 Angstrom resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-phosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final X-factor of 19.8% (R-free:22.5%). The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction. The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction. Structural determinants, which are likely to be responsible for the inability of the S. cerevisiae enzyme to form icosahedral capsids, will be discussed.
|
|
| 5. |
- Zhang, X F, et al.
(författare)
-
X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 angstrom resolution : Determinants of thermostability revealed from structural comparisons
- 2001
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 306:5, s. 1099-1114
-
Tidskriftsartikel (refereegranskat)abstract
- An open reading frame optimized for expression of 6,7-dimethyl-8-ribityllumazine synthase of the hyperthermophilic bacterium Aquifex aeolicus in Escherichia coli was synthesized and expressed in a recombinant E. coli strain to a level of around 15%. The recombinant protein was purified by heat-treatment and gel-filtration. The protein was crystallized in the cubic space group 123 with the cell dimensions a = b = c = 180.8 Angstrom, and diffraction data were collected to 1.6 Angstrom resolution. The structure was solved by molecular replacement using lumazine synthase from Bacillus subtilis as search model. The structure of the A. aeolicus enzyme was refined to a resolution of 1.6 Angstrom. The spherical protein consists of 60 identical subunits with strict icosahedral 532 symmetry. The subunit fold is closely related to that of the B. subtilis enzyme (rmsd 0.80 Angstrom). The extremely thermostable lumazine synthase from A. aeolicus has a melting temperature of 119.9 degreesC. Compared to other icosahedral and pentameric lumazine synthases, the A. aeolicus enzyme has the largest accessible surface presented by charged residues and the smallest surface presented by hydrophobic residues. It also has the largest number of ion-pairs per subunit. Two ion-pair networks involving two, respectively three, stacking arginine residues assume a distinct role in linking adjacent subunits. The findings indicate the influence of the optimization of hydrophobic and ionic contacts in gaining thermostability.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
|
|
