The biosynthetic genes for prenylated phenazines are located at two different chromosomal loci of Streptomyces cinnamonensis DSM 1042.
Bottom Line: The protein EpzP was expressed in Escherichia coli in form of a his-tag fusion protein and purified.The enzyme catalysed the prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dihydro-PCA) using dimethylallyl diphosphate (DMAPP) as isoprenoid substrate.K(m) values were determined as 108 µM for dihydro-PCA and 25 µM for DMAPP.
Affiliation: Eberhard-Karls-University of Tübingen, Pharmaceutical Institute, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.Show MeSH
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Mentions: For the expression and purification of EpzP, its structural gene was again amplified by PCR and cloned into two different expression vectors, pET28a and pHis8. The correct sequence was confirmed for the insert of both constructs and the protein was expressed in E. coli as a fusion protein with an N‐terminal His6 or His8 tag respectively. Both constructs generated equal amounts of protein, and the activity of both fusion proteins were very similar. Ni2+ affinity chromatography resulted in a protein of apparent homogeneity. When this protein was incubated with 5,10‐dihydrophenazine‐1‐carboxylic acid (dihydro‐PCA) and dimethylallyl diphosphate (DMAPP) as substrates, the enzyme‐dependent formation of a single prenylated product was observed (Fig. 5A). The instable prenylated dihydro‐PCA was oxidized to the stable endophenazine A using sodium peroxodisulfate, and the identity of the resulting compound to endophenazine A was confirmed by HPLC‐UV and HPLC‐ESI‐MS in comparison with an authentic reference sample. Both compounds gave identical fragmentation patterns in mass spectrometry.
Affiliation: Eberhard-Karls-University of Tübingen, Pharmaceutical Institute, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.