Design of the enzymatic synthesis
The well-established metabolic pathway for de novo purine biosynthesis is outlined in . Enzymes of the pentose phosphate pathway convert glucose to PRPP, which enters into the linear cascade of reactions that assemble the purine ring to produce IMP, which is the precursor for both ATP and GTP. The net reaction for biosynthesis of ATP and GTP is shown in , including the precursor molecules as well as the numerous cofactors that are consumed in the reaction cascade. The atoms in the purine ring are metabolically derived from glycine, CO2, aspartate, glutamine, and 10-formyl THF as shown in , and this set of precursors defines the possible isotope labeling patterns that can be achieved using in vitro biosynthesis. The goal of the present work is to recapitulate the de novo purine biosynthesis cascade in vitro, using purified recombinant enzymes and a defined set of labeled precursors to generate specifically isotopically labeled nucleotides.
Scheme for Enzymatic Synthesis of Purine Nucleotides
Figure 2 Substrates and products of the biosynthesis for: (a) ATP, (b) GTP. Metabolic origin of purine atoms for: (c) adenine, (d) guanine. The substrates and the metabolic source for each purine atom is color coded: Glycine (blue), CO2 (orange), Aspartate (green), (more ...)
For efficient enzymatic synthesis in vitro, there are three major considerations. First, isotopically labeled amino acids are expensive starting materials. 13C-labeled 10-formyl THF is not commercially available, and it is necessary to generate these labeled precursor components in situ. Second, the numerous ATP and GTP equivalents required cannot be supplied in stoichiometric amounts to avoid isotopic dilution of the desired nucleotide products, and it is essential to employ enzymatic cofactor regeneration schemes. Third, it is important to provide a driving force for the overall reaction to ensure high yields of the desired labeled ATP and GTP. All of these considerations can be taken into account to design and effect an efficient and cost-effective enzymatic synthesis scheme.
The overall strategy for de novo
purine nucleotide synthesis builds on the previously implemented syntheses of nucleotides from glucose and bases, via the PRPP intermediate, as shown in (15
). The cofactor recycling schemes for regeneration of NTPs from NMPs or NDPs and regeneration of NAD(P)+
from NAD(P)H are well-established (34
), shown in . Creatine phosphate is a suitable phosphate source for re-phosphorylation of NDPs by the action of creatine kinase. NAD(P)+
regeneration is accomplished by the action of glutamate dehydrogenase on α-ketoglutarate and ammonia, to produce glutamate. Since neither glutamate nor creatine directly supply labeled atoms to the final product, these reagents may be used in excess to provide a driving force for the overall reaction.
During the ATP synthesis, the enzymes continue to utilize and regenerate labeled ATP as it is produced. To initiate the reaction, it is necessary to add a catalytic amount of unlabeled ATP (1%). In addition, a catalytic amount of unlabeled GTP (1%) is added as a required cofactor for conversion of IMP to adenylosuccinate by adenylosuccinate synthase (purA). The labeled ATP that is synthesized will unavoidably contain 1% unlabeled ATP and 1% unlabeled GTP, but for preparation of labeled RNAs for NMR studies, this has a negligible effect on the overall labeling.
GTP synthesis is equally dependent on ATP, but addition of 1% catalytic ATP is not sufficient to initiate the reaction. Although increasing the ATP concentration is effective, the subsequent separation of labeled GTP from unlabeled ATP involves a difficult chromatography step. Fortunately, the ATP requirement for the GTP synthesis reaction can be met by the addition of 10% dATP that is readily separated from the final product GTP during the boronate affinity chromatography step. Several enzymes including hexokinase, PRPP synthase (prsA), both monophosphate kinases (plsA, spoR) and glutamine synthase (glnA) utilize dATP with slightly lower efficiency than ATP. However, GMP synthase (guaA), will not accept dATP as a substrate, and it is still necessary to add a catalytic amount of ATP (1%) during GTP synthesis in the presence of dATP. Both ATP and dATP are regenerated by creatine phosphokinase, efficiently fueling the GTP reaction. In this way, dATP provides the critical phosphate source in the GTP synthesis reaction without contributing to isotopic dilution or mixing of the nucleotide pool.
To implement the purine biosynthesis cascade, shown in , several additional cofactor recycling schemes needed to be developed. Four of the nitrogen atoms in ATP or GTP are derived from the α-amino group of aspartate or the ε-amino group of glutamine, and both of these positions can be enzymatically labeled, in situ from 15N-ammonium ions, as shown in . Generation of ε-15N-glutamine can be accomplished in situ from glutamate using glutamine synthase, while generation of α-15N-aspartate can be accomplished in situ from fumarate using aspartate-ammonia lyase (aspA). Glutamine is used in three reactions for the incorporation of the purine N3 and N9 atoms and the guanine N2 atom, releasing glutamate, which can be recycled to form ε-15N-glutamine by the action of glnA, as shown in . Aspartate is used in two similar two-step reaction sequences for incorporation of the purine N1 atom and the adenine N6 atom, both reactions releasing fumarate, which can be recycled to form α-15N-aspartate by the action of aspA, as shown in . In this reaction scheme, glutamate is generated in situ from recycling of NAD(P)H, and only catalytic amounts of fumarate must be added. The action of glnA and aspA effectively recycles the pools of glutamine and aspartate cofactors.
The purine C2 and C8 positions are derived from 10-formyl-THF, however 13C-labeled folates are not commercially available. It is possible to generate 13C-10-formyl-THF in situ and recycle catalytic amounts of tetrahydrofolate (THF) as a cofactor, using the scheme shown in . The β-carbon of serine is incorporated into 10-formyl-THF by the sequential action of serine hydroxymethyl transferase (glyA) and the multifunctional enzyme folD, which provides for efficient recycling of the folate cofactor pool during nucleotide synthesis.
The recycling schemes shown in for the NTP, NAD(P)H, glutamate, fumarate, and folate pools, make the overall synthesis of ATP and GTP, shown in , streamlined and efficient. Almost every step of the linear sequence from glucose to ATP/GTP is facilitated by recycling of one of these cofactor pools. The net reaction for the enzymatic scheme shown in including the effects of cofactor recycling, is given in for ATP and GTP synthesis, respectively. The set of starting materials for the overall reaction shown in is simplified to a small number of reagents that fall into three classes. First, there is a set of stoichiometric reagents that are ultimately incorporated into the nucleotide, which includes glucose, CO2, ammonia, and serine. Second, the large number of phosphate and reducing equivalents are supplied with creatine phosphate and α-ketoglutarate, respectively. These two reagents are supplied in excess to provide a driving force that fuels the overall reactions. Third, a set of cofactors is supplied in catalytic amounts. The complex cascade of reactions in is effectively reduced to a net transformation of glucose, CO2, ammonia, and serine into ATP/GTP, with one byproduct molecule of glycine in addition to the creatine and glutamate byproducts from cofactor regeneration.
Figure 3 Pathway engineered synthesis scheme for: (a) ATP and (b) GTP. Reagents are color coded as: Stoichiometric isotopically labeled reagents (black), Phosphate and oxidizing equivalents as the driving force (red), and Recycled cofactors (blue). Cofactors are (more ...)
In the net reaction, carbons of the purine ring originate from only serine (C2,C4,C5,C8) and CO2 (C6), while the nitrogens of the purine ring are derived from serine (N7) and NH4+ ions (N1,N2,N3,N6,N9). There is a tremendous flexibility available generating specific labeling patterns by the suitable choice of labeled starting materials. Due to the availability of specifically labeled serine, all the purine base carbons can be separately labeled except for C2 and C8. For nitrogen, it is possible to separate the N3, N9 from N1 by using labeled aspartate directly, and N7 is separately labeled from serine.
There is one restriction placed on possible labeling patterns due to the production of CO2
by decarboxylation of 6-phosphogluconate to ribulose-5-phosphate during PRPP generation. This step effectively couples the isotope composition of the C1 of glucose to C6 in the purine base via the CO2
pool, and care must be taken to avoid isotopic scrambling or isotopic dilution due to atmospheric CO2
in certain cases. If 13
C-ribose and 13
C6-purine labeling are both desired, then degassing and inert atmosphere are sufficient to prevent isotopic dilution (see Supplementary Figure 9
). If 13
C6 purine labeling is desired without ribose labeling, then PRPP can be generated enzymatically directly from unlabeled ribose (15
). If 13
C-ribose labeling from 13
C-glucose is desired without 13
C6-purine labeling, then an excess of 12
can be supplied. For synthesis of two different labeled nucleotides, 13
C labeling of the base and the ribose moieties was successfully combined.