Microcystins are hepatotoxic, non-ribosomal peptides produced by several genera of freshwater cyanobacteria. Among other enzymic activities, in particular those of peptide synthetases and polyketide synthases, microcystin biosynthesis requires racemases that provide D-aspartate and D-glutamate. Here, we report on the cloning, expression and characterization of an open reading frame, mcyF, that is part of the mcy gene cluster involved in microcystin biosynthesis in the Microcystis aeruginosa strain PCC 7806. Conserved amino acid sequence motifs suggest a function of the McyF protein as an aspartate racemase. Heterologous expression of mcyF in the unicellular cyanobacterium Synechocystis PCC 6803 yielded an active His(6)-tagged protein that was purified to homogeneity by Ni(2+)-nitriloacetate affinity chromatography. The purified recombinant protein racemized in a pyridoxal-5'-phosphate-independent manner L-aspartate, but not L-glutamate. Furthermore, we have identified a putative glutamate racemase gene that is located outside the mcy gene cluster in the M. aeruginosa PCC 7806 genome. Whereas homologues of this glutamate racemase gene are present in all the Microcystis strains examined, mcyF could only be detected in microcystin-producing strains.
Amino Acid Isomerases Amino Acid Sequence