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. 2017 Mar 17;19(6):1426-1429.
doi: 10.1021/acs.orglett.7b00355. Epub 2017 Feb 24.

Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A

Yeonjin Ko  1 , Shao-An Wang  1 , Yasushi Ogasawara  2 , Mark W Ruszczycky  2 , Hung-Wen Liu  1   2
Affiliations

Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A

Yeonjin Ko et al. Org Lett. .

Abstract

Genome scanning of Streptomyces kaniharaensis, the producer of formycin A, reveals two sets of purA, purB, purC, and purH genes. The Pur enzymes catalyze pyrimidine assembly of purine nucleobases. To test whether enzymes encoded by the second set of pur genes catalyze analogous transformations in formycin biosynthesis, formycin B 5'-phosphate was synthesized and shown to be converted by ForA and ForB to formycin A 5'-phosphate. These results support that For enzymes are responsible for formycin formation.

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Figures

Figure 1
Figure 1
Biosynthetic origins of formycin A and adenosine.
Figure 2
Figure 2
In vitro assay of ForA and ForB. (A) Reaction mixture containing formycin B 5′-phosphate (12b) without enzymes; (B) 12b + ForA; (C) 12b + ForA + ForB. Reaction mixtures for traces (A) to (C) were analyzed after a 1.5 h incubation by HPLC using a Dionex CarboPac PA1 anion exchange column in a two-solvent system of H2O versus 1.5 M ammonium acetate in H2O (pH 7.0). (D) Isolated ForA/ForB product; (E) isolated ForA/ForB product + CIP; (F) coinjection of isolated ForA/ForB/CIP product and formycin A (1) standard. HPLC traces (D) to (F) were obtained using a Microsorb-MV 100-5 C18 column in a two-solvent system of 1% ammonium acetate in H2O (pH 5.1) versus acetonitrile (see Supporting Information and text for details).
Figure 3
Figure 3
Competition assay of ForA with 12a and 12b. (A) Sample HPLC traces at three time points from a single trial showing relative consumption of 12b versus 12a. HPLC traces utilized a Microsorb-MV 100-5 C18 column with a two-solvent system of 1% ammonium acetate in H2O (pH 5.1) versus acetonitrile. (B) Plots of log([12a]/bi) versus log([12b]/a) for the five separate trials where bi and a are scaling factors relating the concentrations to the standardized HPLC peak integrations and shift the intercepts to facilitate visualization (see text and Supporting Information for details).
Scheme 1
Scheme 1
The later steps of the established biosynthetic pathway for adenosine (2) and the proposed analogous pathway for formycin A (1) formation.
Scheme 2
Scheme 2
Chemical synthesis of formycin B 5′-phosphate (12b).
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