Biosynthetic versatility and coordinated action of 5'-deoxyadenosyl radicals in deazaflavin biosynthesis - PubMed (original) (raw)

. 2015 Apr 29;137(16):5406-13.

doi: 10.1021/ja513287k. Epub 2015 Apr 20.

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Biosynthetic versatility and coordinated action of 5'-deoxyadenosyl radicals in deazaflavin biosynthesis

Benjamin Philmus et al. J Am Chem Soc. 2015.

Abstract

Coenzyme F420 is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F(o)) is still poorly understood. F(o) synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5'-deoxyadenosyl radicals to catalyze F(o) formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a p-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F(o) synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5'-deoxyadenosyl radicals to mediate the formation of a complex heterocycle.

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Figures

Figure 1

Figure 1

Biosynthesis of the deazaflavin chromophore of F420 (Fo, 3). The structure of F420 shown contains a single glutamic acid, which is designated as F420-1. The number of glutamic acid residues varies.

Figure 2

Figure 2

Mechanistic proposal for the Fo-synthase-catalyzed reaction. AdG and AdH correspond to the 5′-deoxyadenosyl radicals generated at the CofG and the CofH active sites by reduction of SAM using the active site [4Fe-4S]+1 cluster., Abstraction of the tyrosine amine hydrogen atom by the CofH 5′-deoxyadenosyl radical gives 5, which then undergoes fragmentation leading to the formation of the _p_-hydroxybenzyl radical 7. This then undergoes addition to the double bond of diaminouracil 2 followed by oxidation to give 9. Transfer of 9 from the CofH active site to the CofG active site gives 10. Hydrogen atom abstraction, by the CofG 5′-deoxyadenosyl radical, gives 11. Cyclization to 13, followed by oxidation by the [4Fe-4S]+2 cluster and elimination of ammonia completes the formation of the deazaflavin 3.

Figure 3

Figure 3

Deuterium incorporation into 5′-deoxyadenosine (left spectrum, [M + H]+ = 252.1 Da) and Fo (right spectrum, [M + H]+ = 364.1 Da, [M + K]+ = 402.1 Da) from various isotopologues of tyrosine: (a) tyrosine, (b) D7-tyrosine, or (c) 3,3-D2-tyrosine. The percentages written above the 253.1 Da peak indicate the relative height compared to the 252.1 peak. Each reaction mixture contained methyl viologen, SAM, diaminouracil, D_n_-tyrosine, CofH, and CofG.

Figure 4

Figure 4

CofH-catalyzed deuterium incorporation into 5′-deoxyadenosine in reactions run in 80% D2O containing SAM, diaminouracil and methyl viologen. (a) Reaction run in the presence of tyrosine showing an enhanced 253.1 peak (calculated to be 69.4, 21.5 and 16.7% for the [M + D + H]+, [M + D2 + H]+, [M + D3 + H]+, respectively, in panel a and 30.8% for the [M + D + H]+ in panel b). (b) Reaction run in the absence of tyrosine showing the uncoupled production of 5′-deoxyadenosine. 5′-deoxyadenosine has an expected [M + H]+ of 252.1 m/z with the 253.1 peak calculated to be 12.9 ± 3%.

Figure 5

Figure 5

Trapping of the tyrosine-derived glyoxylate 15. (a) Proposed scheme for the generation of glyoxylate 15 by hydrolysis of the glycine imine 6 and its trapping using _o_-phenylenediamine 16. (b) Extracted ion chromatograms at m/z 148.05 ± 0.1 of labeled 2-quinoxalinol 17 generated from a reaction mixture containing CofG + CofH + SAM + sodium dithionite + [15N,13C9]-tyrosine + 2 (red trace); CofH + SAM + sodium dithionite + [15N,13C9]-tyrosine + 2 (blue trace). Control reactions lacking SAM, sodium dithionite, tyrosine, or CofH did not produce labeled glyoxylate.

Figure 6

Figure 6

Detection of the CofH reaction product. (a) Reaction catalyzed by CofH. (b) HPLC chromatogram of the reaction mixture containing CofH + SAM + methyl viologen (reduced) + 1 + 2 (red) showing a new product eluting after 11.15 min. Reaction mixtures lacking reduced methyl viologen (green), SAM (black), or tyrosine (blue) did not show this product.

Figure 7

Figure 7

MS analysis of the CofH reaction product. (a) Mass spectrum of the product generated from tyrosine ([M + H]+ obs. 383.1573, calcd for 9, 383.1561, 3.3 ppm error, [M + K]+ obs. 421.1132, calcd for 9, 421.1120, 2.8 ppm error; (b) mass spectrum of the product generated from [15N,13C9]-Tyr ([M + H]+ obs. 390.1775, calcd for [13C7]-9, 390.1796, 5.4 ppm error, [M + K]+ obs. 428.1322, calcd for 9, 428.1355, 7.7 ppm error); (c) MS2 of the product from tyrosine (m/z 383.1); (d) MS2 of the product from [15N,13C9]-tyrosine (m/z 390.1). The isolation width was 10 m/z and the collision energy was 30 V. (e) Proposed CID fragmentation pattern of the CofH product 9.

Figure 8

Figure 8

Incorporation of deuterium into 5′-deoxyadenosine in reactions consisting of SAM, [5,5-D2]-9 and dithionite reduced CofG. (a) Mass spectrum of 5′-deoxyadenosine (calcd 252.1 with a 253.1 peak calculated to be 12.9 ± 3% relative intensity; (b) mass spectrum of Fo (calcd 364.1). The presence of the 252.1 peak in the 5′-deoxyadenosine MS is most likely due to the uncoupled production of 5′-deoxyadenosine.

Figure 9

Figure 9

Detection of an intermediate produced by FbiC-C1. (a) HPLC analysis (Abs 257 nm) of the reaction mixtures containing SAM + tyrosine (1) + diaminouracil (2) + flavodoxin/flavodoxin reductase in the presence of FbiC (wild-type) (25 μM) or variants (FbiC-C1 or FbiC-C2, each at 50 μM). (b) HPLC analysis (Abs 257 nm) of the reaction of FbiC-C1 demonstrating that the production of the intermediate is dependent on both tyrosine (1) and diaminouracil (2).

Figure 10

Figure 10

Fo production by FbiC-C1 and FbiC-C2 variants compared to FbiC-wt. The FbiC proteins were incubated with SAM, tyrosine (1) and diaminouracil (2), and the reaction was initiated with the flavodoxin/flavodoxin reductase system. The estimated rate of FbiC-catalyzed Fo formation is 2.4 × 10–5 s–1.

Figure 11

Figure 11

Stereochemistry of deuterium transfer from C3 of tyrosine during CofG/CofH-catalyzed Fo formation. (a) Mass spectrum of Fo derived from [3,3-D2]-Tyr; (b) mass spectrum of Fo derived from [2,3-D2, 3_S_]-Tyr (1c); (c) mass spectrum of Fo derived from [3-D, 3_R_]-Tyr (1d). (Fo calcd [MH + H]+ 364.1, calcd [MD + H]+ 365.1, calcd [MH + K]+ 402.1, calcd [MD + K]+ 403.1.

Figure 12

Figure 12

Comparison of the reaction catalyzed by ThiH (thiamin biosynthesis) HydG (FeFe hydrogenase biosynthesis), NosL (Nosiheptide biosynthesis), and the proposed reaction catalyzed by CofH (F420 biosynthesis). Reversible abstraction of the exchangeable hydrogen atom has been observed for ThiH, (Begley,T. P. and Mehta A, unpublished) HydG, and CofH and structural studies on NosL clearly demonstrates abstraction of the amino hydrogen.

Figure 13

Figure 13

Strategy to detect β bond cleavage involving loss of stereochemical information at C3 of tyrosine.

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