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1.  Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers 
BMC Biochemistry  2005;6:24.
Background
Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15N/13C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-13C6]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15N-phenylalanine.
Results
Quantitative NMR analysis showed incorporation of the proffered [U-13C6]glucose into the ribose moiety of ribonucleosides (40 – 45%) and into the amino acids, alanine (41%), glutamic acid/glutamine (C-4 and C-5, 30%) and aspartate/asparagine (15%). Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%). Prior to the incorporation into protein the proffered 15N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent.
Conclusion
Growth of S. frugiperda cells in the presence of [U-13C6]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15N-labelled amino acids may be hampered by loss of the 15N-label by transamination.
doi:10.1186/1471-2091-6-24
PMCID: PMC1310531  PMID: 16285881
2.  Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments 
BMC Biochemistry  2003;4:18.
Background
Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.
Results
A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.
Conclusion
Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.
doi:10.1186/1471-2091-4-18
PMCID: PMC337094  PMID: 14690539

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