A key cytochrome P450 hydroxylase in pradimicin biosynthesis

Abstract

Pradimicins A-C (1–3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A D-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115 μM under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.

Synergistic Actions of Tailoring Enzymes in Pradimicin Biosynthesis

“…[17] The relaxed amino acid substrate specificity of PdmN makes it a useful biocatalyst to generate novel pradimicin analogues. Previous attempts to express PdmN in E. coli BL21 (DE3) using the expression vector pET28a failed because an insoluble form of the enzyme was generated [18] and similar results were obtained with other expression vectors such as pACYC Duet-1 and with different E. coli expression conditions. In the current work, PdmN expression in S. coelicolor CH999 was attempted since functional expression of PdmN and other biosynthetic enzymes was successful to afford 4 .…”

“…The conversion rate was improved when glucose dehydrogenase was co-expressed to recycle the co-factors. [18] Although both PdmJ and PdmW can cytalyze the corresponding hydroxylations of 3 , the efficienties of both individual reactions were low. However, these two CYP enzymes work collaboratively to efficiently generate a new pradimcin bisynthetic intermediate 7 .…”

“…Our previous work on PdmJ revealed substrate inhibition of this enzyme in the in vitro reactions. [18] The synergistic actions of PdmJ and PdmW are likely due to the prevention of substrate inhibition in pradimicin biosynthesis.…”

“…This is consistent with the in vitro results from our previous work. [18] In contrast, when pKN102 that harbors pdmABCDGHKLNW was expressed in S. coelicolor CH999, several products including 3 , 4 , 6 , and 8 (0.3± 0.1 mg L −1 ) were biosynthesized (trace iv, Figure 5). The UV spectrum of 8 suggested it is a pradimicin analogue (Figure S1).…”

“…Hydroxylation of 3 by PdmJ yielded JX134 ( 5 , Scheme 1). [17, 18] This hydroxyl group serves as a site for the subsequent glycosylations. Given the essential roles of the D-alanine and sugar moieties in the biological activities of 1 , it is important to understand the catalytic properties of these tailoring enzymes and how they work together in pradimicin biosynthesis.…”