Textbooks and metabolic maps often give the mistaken impression that all of the pathways of intermediary metabolism are known. Actually, there are many well-known biomolecules for which the biosynthetic pathways are poorly understood or never studied. For example, the origin of the eight-carbon atoms comprising the xylene-ring of riboflavin was deduced as recently as 1983 (1). The classical approach using radiolabeled precursors required complicated chemical degradation procedures to locate labeled atoms. This tedious and difficult procedure, once worthy of Nobel prizes (eg. Bloch 1964, Cholesterol biosynthesis), has given way to a much preferred tracer strategy.

Carbon 13 is a non-radioactive isotope of carbon. In contrast to carbon 12, this isotope has a nuclear spin that can be detected by nuclear magnetic resonance (NMR). Because the chemical environment of an atomic nucleus influences it chemical shift in NMR, most if not all carbon atoms in an organic molecule can be identified nondestructively. Furthermore, the natural abundance of ¹³C is only 1.1%, thus ¹³C-enrichment from various precursors can be detected easily in an isolated product.

All of the resonances in the ¹³C-NMR spectrum of riboflavin have been assigned and are indicated in the table below. Also shown are the relative ¹³C-enrichment for each carbon atom of riboflavin after feeding the indicated ¹³C-precursors to the mold, Ashbya gossypii. The researchers purified the riboflavin and acetylated the four hydroxyl groups of the ribityl side chain of riboflavin before analysis, which provided an internal standard for ¹³C natural abundance. As with any experiment of this type, there will be dilution of the isotope by endogenous precursors and product.

Relative ¹³C-enrichments of riboflavin tetraacetates obtained from various ¹³C-labeled precursors.
 

Carbon Atom  in Riboflavin	Chemical Shift ppm	Relative Enrichments
		[1-13C] Acetate	[2-13C] Acetate	[1-13C] Ribose	[1-13C] Glucose	[6-13C] Glucose	[2-13C] Glycerol
2	154.9	1.8	2.7	0.9	1.1	1.6	5.4
4	159.5	4.9	1.9	0.8	1.2	1.2	1.4
4a	136.0	0.9	0.6	0.9	1.3	1.1	1.9
5a	136.6	6.3	5.2	3.3	2.4	2.2	15.9
6	132.8	3.3	5.8	10.3	5.5	3.2	12.9
7	137.1	8.3	3.0	0.8	1.4	1.6	3.0
7a	19.5	1.2	7.0	0.9	3.3	9.0	2.4
8	148.1	5.5	3.6	3.7	1.8	1.9	12.1
8a	21.5	3.3	6.0	9.1	5.0	2.7	12.3
9	115.6	1.2	6.4	1.1	3.4	9.0	2.1
9a	131.2	8.5	2.1	0.6	1.2	1.6	3.0
10a	150.6	1.3	0.8	0.6	0.7	0.9	1.1
1'	45.0	3.2	4.6	19.4	4.5	2.3	8.5
2'	69.4	4.4	4.0	2.9	1.8	1.6	7.4
3'	70.4	6.9	2.7	0.6	1.5	1.7	2.1
4'	69.0	1.0	4.9	1.3	1.9	1.9	12.9
5'	61.9	1.1	4.7	1.1	3.2	6.8	1.6
CH3CO	20.4-21.1	1.0	1.0	1.0	1.0	1.0	1.0
CH3CO	169.8-170.7	1.0	0.8	0.9	0.9	0.8	0.9
The final two entries are from the four acetyl groups on the tetraacetylated riboflavin.  The values are the average of the four signals and serve as a internal, natural abundance standard.

 

Questions:

1.  GTP is a precursor of riboflavin. Draw the structure of GTP next to riboflavin so that the structural similarities of corresponding atoms can be readily appreciated.  Try to deduce which atoms of riboflavin are derived from GTP and which are added.

2.  Examine the ¹³C-labeling pattern for riboflavin from the various labeled precursors. Is the labeling pattern of those carbons derived from GTP consistent with the known biosynthetic pathway for GTP? Justify your answer.

3.  Using circles, squares, triangles, etc. or different colors drawn on the structure above, identify carbon atoms in riboflavin that have a common pattern (origin).

4.  Based on the distribution of label in your answer to Question 3, what can you deduce about the biosynthesis of the xylene ring moiety of riboflavin?

5.  Considering the coupling of nuclear spins detectable by NMR, what biosynthetically important information could be obtained by comparing the ¹³C-NMR spectra of riboflavin synthesized from [4-¹³C] or [5-¹³C] Ribose versus that synthesized from [4,5-¹³C] Ribose? Here [4,5-¹³C] Ribose is doubly labeled, not a mixture of [4-¹³C] and [5-¹³C] Ribose.)

(1) Bacher A, Le Van Q, Keller PJ, Floss HG. (1983) Biosynthesis of riboflavin. Incorporation of ¹³C-labeled precursors into the xylene ring. J Biol Chem. 258(22),13431-7.