Figure 1.
Prenylation in the biosynthesis of phenazines.
(A) Reaction catalyzed by the previously discovered prenyltransferases PpzP and EpzP. (B) Structures of the phenazines JBIR-46, -47, and -48 from Streptomyces sp. SpC080624SC-11.
Figure 2.
Phenazine biosynthesis in Streptomyces sp. SpC080624SC-11.
(A) Putative biosynthetic gene cluster of JBIR-46, -47, and -48. (B) Proposed pathway for the biosynthesis of JBIR-46, -47, and -48. See Table 1 for additional information on the genes contained in the depicted gene cluster.
Table 1.
Genes in the putative biosynthetic gene cluster for JBIR-46, -47, and -48.
Figure 3.
HPLC and LC-MS analysis of the reaction products of Mpz10.
(A) and (B): Incubations containing 1,6-dihydroxyphenazine, dimethylallyl diphosphate (DMAPP), and Mg2+ with the membrane fractions of E. coli harboring either the empty vector pET-28a(+) (A) or the Mpz10 expression vector pPH23 (B). (C) Authentic DHDMP (structure see Figure 2B). (D) and (E): Authentic JBIR-47 and JBIR-46 (structures see Figure 1B). (F) Incubation containing 1-hydroxyphenazine, DMAPP, and Mg2+ with the membrane fraction of E. coli harboring the Mpz10 expression vector pPH23 (for the structure of 3 see Figure 5). Mass spectra of the three enzymatic products are shown on the right. Detection: A-E, UV 275 nm; F, UV 368 nm.
Table 2.
Localization of the Mpz10 activity in the membrane fraction.
Figure 4.
Biochemical investigation of the Mpz10 reaction.
(A) HPLC analysis of the time-dependent formation of products 1 and 2. Detection: UV, 275 nm. (B) and (C): Product formation at different concentrations of 1,6-dihydroxyphenazine and dimethylallyl diphosphate (DMAPP). In the experiments shown in (B), DMAPP was kept constant at 0.5 mM. In the experiments shown in (C), 1,6-dihydroxyphenazine was kept constant at 0.2 mM. Km values were determined by nonlinear regression, using GraphPad Prism software. Data represent mean ± SD of triplicate determinations.
Figure 5.
Structure of the enzymatic product 1-hydroxy-4-dimethylallyl-phenazine (3).
Bold lines indicate key 1H-1H COSY and arrows key 1H-13C HMBC correlations.
Table 3.
NMR spectroscopic data for 1-hydroxyphenazine and the enzymatic product 3 (structure see Figure 5).