Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis

Abstract

The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA incubated with GTP, S-adenosyl-L-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3',8-cyclo-7,8-dihydroguanosine 5'-triphosphate (3',8-cH2GTP). The isolated 3',8-cH2GTP was converted to cPMP by MoaC or its human homologue, MOCS1B, with high specificities (Km < 0.060 μM and 0.79 ± 0.24 μM for MoaC and MOCS1B, respectively), suggesting the physiological relevance of 3',8-cH2GTP. These observations, in combination with some mechanistic studies of MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C-C bond formation reaction and that, in contrast to previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.

Figure 1

(a) Moco biosynthetic pathway in bacteria and humans. The human enzymes are indicated in parenthesis. The symbols on GTP and cPMP indicate the source of the carbon and nitrogen atoms in cPMP as determined by isotope labeling studies^,. P designates a phosphate group. 3′,8-cH₂GTP is shown in brackets, as it had not been identified prior to this report. (b) Previous proposal for the functions of MoaA and MoaC with 2-amino-5-formylamino-6-ribofuranosylamino- 4-pyrimidinone triphosphate (6) as an intermediate^,.(c) Previous proposal for the functions of MoaA and MoaC by Begley et al.. Pyranopterin triphosphate 7 was proposed as the product of MoaA.

Figure 2. Activity assays of MoaA

(a) HPLC analysis of stepwise MoaA/MoaC activity assay (Ex. 367 nm, Em. 450 nm). In the complete reaction, GTP (1 mM), SAM (1 mM) and sodium dithionite (1 mM) were incubated with MoaA (5 μM) for 60 min at 25 °C, followed by removal of MoaA by ultrafiltration and incubation with MoaC (15 μM) for 60 min at 25 °C. cPMP was converted tocompound Z (8), and detected by HPLC. Also shown are the chromatograms for the compound Z standard, and control reactions without GTP or SAM. (b) HPLC analysis of MoaA activity assay (Ex. 365 nm, Em. 445 nm). In the complete condition, MoaA (5 μM), was incubated with GTP (1 mM), SAM (1 mM) and sodium dithionite (1 mM) at 25 °C for 60 min. The reaction product was subjected to acid hydrolysis, followed by incubation with 2,3-butanedione, and analyzed for the fluorescent DMPT (9). Also shown are the chromatograms for the DMPT standard, and for control reactions lacking GTP, SAM, dithionite, or MoaA. A small amount of DMPT (~10% of the complete condition) was formed in the GTP negative control due to the co-purification of GTP with MoaA. (c) Time-course of 3′,8-cH₂GTP formation determined after conversion to DMPT (filled circles) or compound Z (open circle). The conditions for the MoaA reaction and the derivatization of the product to DMPT are identical to (b). The amount of compound Z at 60 min is based on the quantitation of the HPLC peak in (a). Each point is an average of three replicates, and the error bars are calculated based on the standard deviation.

Figure 3

(a) Stability of 3′,8-cH₂GTP under different conditions. Solution containing 3′,8-cH₂GTP was incubated for 10 - 180 min under specified conditions. At each time point, an aliquot was removed and 3′,8-cH₂GTP was quantified by HPLC after its conversion to compound Z. Each point is an average of three replicates, and the error bars are calculated based on the standard deviation.(b) UV-vis absorption spectrum of 3′,8-cH₂GTP (bold trace) (60 μM). The thin traces are spectra after exposure to air at 22 °C for the specified time.

Figure 4. NMR structural characterization of 3′,8-cH₂GTP

(a) ¹H NMR (600 MHz, D₂O) and (b) ¹H-decoupled ¹³C NMR (200 MHz, D₂O) spectra of 3′,8-cH2GTP and [U-₁₃C₁₀^,15N₅]3′,8-cH₂GTP, respectively. No signal associated with 3′,8-cH₂GTP was observed outside the shown chemical shift range. The intensity of the H-1′ signal in (a) was decreased due to the water suppression at 4.95 ppm. The insets in (b) shows magnified view of the signals for C-3′, C-4′ and C-8. (c) ¹³C-¹³C COSY spectrum of [U-¹³C₁₀^,15N₅]3′,8-cH₂GTP. The assignments for the observed correlations are indicated. The chemical shift region for C-1′ - C-5′, and C-8 is shown. The spectrum with full chemical shift range is available in Figure S6. (d) ¹³C-¹³C COSY (bold), and selected ¹H-¹³C, and ¹H-¹⁵N HMBC (single-headed arrows) correlations, and NOE (a double-headed arrow). A complete list of the observed HMBC correlations is available in Table 1. P designates a phosphate group.

Figure 5. Mechanistic studies on the MoaA catalyzed H-atom abstraction.<

br>(a) Stoichiometry of formation of 5′-dA (open circles) and cPMP (filled circles) in the MoaA/MoaC coupled assay. MoaA (20 μM) was incubated with GTP (1 mM), SAM (1 mM) and sodium dithionite (1 mM) in the presence of MoaC (40 μM) at 25 °C. cPMP was quantified by HPLC after its conversion to compound Z. Each point is an average of 3–6 replicates, and the error bars are calculated based on the standard deviation. (b) Rate of cPMP formation from GTP or [3′-²H]GTP by MoaA and MoaC. MoaA (0.5 μM) was incubated with GTP or [3′-²H]GTP (0.1 mM) in the presence of sodium dithionite (1 mM), SAM (1mM) and MoaC (5 μM) at 25°C. cPMP was quantified by HPLC after its conversion to compound Z. Each point is an average of four replicates, and the error bars are calculated based on the standard deviation. (c) ESI-TOF-MS and (d) ¹H NMR (600 MHz in D₂O) and 2H NMR (76.75 MHz in H₂O) spectra of 5′-dA isolated from the reaction in the presence of MoaA (50 μM), MoaC (100 μM), SAM (1 mM), [3′-²H]GTP (98 μM, 94 ± 3 % atom 2H) and sodium dithionite (1 mM). The signal indicated with (*) is non-deuterated 5′-dA (m/z [M+H]⁺ calcd. for C₁₀H₁₄N₅O₃ 252.110; found 252.111) presumably derived from non-labeled GTP (see main text).

Figure 6

Proposed mechanism for the MoaA catalyzed conversion of GTP to 3′,8-cH₂GTP.

Figure 7. A model of the MoaA active site

SAM was modeled into the X-ray crystal structure of MoaA in complex with GTP (PDB ID: 2FB3) based on the structural alignment with the X-ray crystal structure of MoaA in complex with SAM (PDB ID: 1TV8). Oxygens are shown in red, nitrogens in blue, sulfurs in yellow, phosphorous in orange, and irons in brown.

Figure 8. Possible mechanism of the MoaC catalyzed conversion of 3′,8-cH₂GTP to cPMP

P designates a phosphate group.