The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumour mithramycin.
Bottom Line: Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells.The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates.All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
Affiliation: Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), Universidad de Oviedo, Oviedo, Spain.Show MeSH
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Mentions: Two sugars (l‐chromose B and one d‐oliose) in 2 are acetylated at the C4 hydroxyl groups (Fig. 1). These acetyl groups are incorporated into the molecule by the chromomycin acetyltransferase CmmA (Menéndez et al., 2004b). Since these groups greatly increase the antitumour activity of 2, we decided to use CmmA in order to generate acetylated derivatives of 1 with high antitumour activity. To achieve this, we performed a bioconversion experiment by growing the wild‐type strain Streptomyces griseus ssp. griseus (chromomycin A3 producer) that was subsequently fed with 1. These cultures were then extracted and analysed by HPLC‐MS. In addition to 1, a new peak was detected showing the same absorption spectrum than 1, but with higher retention time and an m/z value in positive mode of 1128 (data not shown). This is in agreement with compound 1 with one acetyl group attached. We also tested if this acetylation event was CmmA‐dependent by running a bioconversion experiment using S. griseus C10A as host (cmmA‐ mutant). No acetylated mithramycin was obtained in these conditions confirming that CmmA is required for this acetylation process (data not shown). This result showed that CmmA was flexible enough to be able to acetylate 1. The bioconversion efficiency using the wild‐type strain was rather low (approximately 14%). Most probably this was due to the fact that the acetyltransferase CmmA has more affinity to its genuine substrate and/or was mainly committed to the biosynthesis of 2. With the aim of improving the production of acetylated derivatives of 1, we tested the bioconversion efficiencies as biocatalysts of different mutant strains of S. griseus affected in biosynthesis of 2. These mutants (C60GI, C10GII, G10GIII and G10GIV; Fig. 2) expressed the acetyltransferase CmmA but were blocked in glycosylation steps taking place before the acetylation of sugars, and therefore preventing the synthesis of potential natural substrates for CmmA. When any of these mutants was used in bioconversion experiments with 1 as substrate, higher production of monoacetylated mithramycin 11 was obtained. Since S. griseus C10GIV was more efficient than the other mutant strains in the bioconversion experiments (35% of conversion, Fig. 3A), it was selected as biocatalyst for further experiments.
Affiliation: Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), Universidad de Oviedo, Oviedo, Spain.