Enzymatic synthesis and structural characterization of 13C, 15N-poly(ADP-ribose)

Abstract

Poly(ADP-ribose) is a significant nucleic acid polymer involved with diverse functions in eukaryotic cells, yet no structural information is available. A method for the synthesis of (13)C, (15)N-poly(ADP-ribose) (PAR) has been developed to allow characterization of the polymer using multidimensional nuclear magnetic resonance (NMR) spectroscopy. Successful integration of pentose phosphate, nicotinamide adenine dinucleotide biosynthesis, and cofactor recycling pathways with poly(ADP-ribose) polymerase-1 permitted labeling of PAR from (13)C-glucose and (13)C, (15)N-ATP in a single pot reaction. The scheme is efficient, yielding approximately 400 nmoles of purified PAR from 5 mumoles ATP, and the behavior of the synthetic PAR is similar to data from PAR synthesized by cell extracts. The resonances for (13)C, (15)N-PAR were unambiguously assigned, but the polymer appears to be devoid of inherent regular structure. PAR may form an ordered macromolecular structure when interacting with proteins, and due to the extensive involvement of PAR in cell function and disease, further studies of PAR structure will be required. The labeled PAR synthesis reported here will provide an essential tool for the future study of PAR-protein complexes.

Figure 1

Chemical structure of poly(ADP-ribose).

Scheme 1. PAR Synthesis using nicotinamide recycling and NAD⁺ biosynthesis

Poly(ADP-ribose)_n and NAD⁺ (1) are converted to poly(ADP-ribose)_n+1 by the action of poly(ADP-ribose) polymerase 1 (PARP-1) with the production of nicotinamide. Nicotinamide (2) and water are converted to nicotinate by the action of nicotinamidase (pncA) with the production of an ammonium ion. Nicotinate (3) and phosphoribosyl-pyrophosphate (PRPP) are converted to nicotinate nucleotide by the action of nicotinate phosphoribosyl transferase (pncB) with the production of pyrophosphate. Nicotinate nucleotide (4) and ATP are converted to deamido-NAD⁺ by the action of nicotinate nucleotide adenylyl-transferase (nadD) with the production of pyrophosphate. Deamido-NAD⁺ (5), ATP and glutamine are converted to NAD⁺ by the action of NAD+ synthase (nadE) with the production of glutamate, AMP and pyrophosphate. Glucose and ATP are converted to glucose-6-phosphate by the action of hexokinase (hxkA) with the production of ADP. Glucose-6-phosphate and NADP⁺ are converted to 6-phosphogluconate by the action of glucose-6-phosphate dehydrogenase (zwf) with the production of NADPH. 6-Phosphogluconate and NADP⁺ are converted to ribulose-5-phosphate by the action of 6-phosphogluconate dehydrogenase (gndA) with the production of NADPH. Ribulose-5-phosphate is interconverted to ribose-5-phosphate by the action of phosphoriboisomerase (rpiA). Ribose-5-phosphate and ATP are converted to PRPP by the action of PRPP synthase (prsA) with the production of AMP. ATP and NADP⁺ recycling is achieved by creatine phosphokinase (ckmT), adenylate kinase (adk) and glutamic dehydrogenase (gdhA).

Figure 2. Time course of PAR Synthesis

Denaturing 20% polyacrylamide gel shows time point products from the ¹³C, ¹⁵N- PAR synthesis. PAR, NAD⁺ and ATP are labeled and visualized with ³²P by the addition of a trace amount of α-³²P-ATP to the synthesis reaction.

Figure 3. HSQC spectra for ¹³C, ¹⁵N- poly(ADP-ribose)

¹H-¹³C- correlation including assignments for each resonance is shown for the ribose (A) and aromatic (B) regions of PAR. ¹H-¹⁵N- 2J coupling of adenine nitrogen atoms of PAR is shown in (C). The H8 proton is coupled to N7 and N9, while the H2 proton is coupled to N1 and N3. The dashed line indicates the chemical shift of these protons. An extra resonance at 223 ppm appears as a result of N9 signal folding in.

Figure 4. Through space correlation of protons in Adenosine compounds

(A) Homonuclear ROESY of ADP-ribose monomer. The homonuclear NOESY spectra of poly(ADP-ribose) (B) and poly(Adenosine) (C) are also shown.

Figure 5. Thermal Denaturation of PAR at various concentrations of NaCl

The transition in absorbance at 4 M NaCl suggests that poly(ADP-ribose) is more structured under these conditions.

Figure 6. Comparison of ADP-ribose monomer and polymer at low and high salt

In both cases, the polymer linewidths are broader than those of the monomer, as expected. The increased broadening of signals at 4 M NaCl suggests that PAR may adopt a macromolecular structure.