PMC

Spectra: A, ¹H NMR; B, ³¹C NMR; C, ¹H/¹³C HSQC NMR; D. ¹H/¹³C HMBC NMR.

Protons, carbons and phosphorus of 2NAcAmEtPn are labeled in red. Spectra: A, ¹H NMR; B, ¹³C NMR; C, ¹H/¹³C HSQC NMR; D, ¹H/³¹P HMBC NMR.

Symbols: squares, AmMePn (δ 9.2 ppm); diamonds, NAcAmMePn (δ 13.9 ppm); triangles, Rib1′NAcAmMePn (δ 17.4 ppm).

Symbols: squares, 2AmEtPn (δ 16.8 ppm); diamonds, 2NAcAmEtPn (δ 19.4 ppm); triangles, Rib1′N2AcAmEtPn (δ 23.6 ppm); circles, Rib1,2cP (δ 18.6 ppm). (A) Strain HO2568 (phn⁺ ΔpstS) (closed symbols) and strain HO2541 (phnO38 ΔpstS) (open symbol); (B) strain HO2542 (phnP ΔpstS); (C) strain HO2536 (phnJ ΔpstS).

Black arrows: correlations observed by ¹H/¹³C HSQC and ¹H/¹³C HMBC spectroscopy; blue arrows: correlations observed by ¹H/³¹P HMBC spectroscopy. A, 2NAcAmEtPn; B, 5′PRib1′2NAcAmEtPn.

(A) Reaction mixture without enzyme containing AmMePn (δ 9.2 ppm), 2AmEtPn (δ 17.7 ppm), R1AmEtPn (δ 12.7 ppm) and S1AmEtPn (12.7 ppm). The ³¹P NMR chemical shifts of acetyl coenzyme A (not shown) were δ 1.7 ppm (s), δ −10.7 ppm (d, J = 19 Hz) and δ −11.5 ppm (d, J = 19 Hz). (B) Reaction product (14.3 ppm) after incubation of enzyme, acetyl coenzyme A and AmMePn, (C) reaction product (δ 20.5 ppm) after incubation of enzyme, acetyl coenzyme A and 2AmEtPn, (D) reaction product (δ 18.3 ppm) after incubation of enzyme, acetyl coenzyme A and S1AmEtPn, (E) reaction product (δ 18.3 ppm) after incubation of enzyme, acetyl coenzyme A and R1AmEtPn.

Cells of strain HO2568 (phn⁺ ΔpstS) were grown in 03P medium in the presence of 2AmEtPn and supernatant fluids were analyzed as described in . (A) ³¹P NMR spectrum of culture supernatant immediately after addition of 2AmEtPn. Chemical shifts: 16.8 ppm, 2AmEtPn; 2.3 ppm P_i. (B) ³¹P NMR spectrum of culture supernatant after 20 h of incubation with 2AmEtPn. Chemical shifts: 23.6 ppm, Rib1′N2AcAmEtPn; 19.4 ppm, 2NAcAmEtPn; 2.3 ppm P_i. The peak at 0.0 ppm represents the external standard.

Compounds: 1, NAcAmMePn; 2, 5′-triphospho-α-d-ribosyl 1′-(N-acetamidomethylphosphonate); 3, 5′PRib1′NAcAmMePn; 4, N-methylacetamide; 5, 5PRib1,2cP; 6, α-d-ribosyl 1,5-bisphosphate; 7, PRPP; 8, diphosphate. Reactions are indicated by their enzymes: PhnO, aminoalkylphosphonate N-acetyltransferase; PhnI*, an enzyme complex where PhnI plays a crucial catalytic role, and which may involve also PhnG, PhnH, PhnJ, PhnK and/or PhnL; PhnM, 5′-triphospho-α-d-ribosyl 1′-phosphonate diphosphohydrolase; PhnJ*, S-adenosylmethionine dependent carbon-phosphorus lyase. PhnJ* may constitute a protein complex containing also PhnG, PhnH, PhnI, PhnK and/or PhnL. PhnI* and PhnJ* may be the same protein complex; PhnP, phnP specified phosphoribosyl cyclic phosphodiesterase; PhnN, phnN specified ribosylbisphosphate phosphokinase; APRTase, apt specified adenine phosphoribosyltransferase; Ppa, ppa specified inorganic diphosphate hydrolase. The enzymes of the latter two reactions are not specified by the phn operon. APRTase is arbitrarily chosen among the 10 phosphoribosyltransferases of E. coli . Any of these 10 enzymes may participate in the process. The pathway is established on the basis of refs. 4, 5, 6, 7, 11 and 13, as well as results of the present work.