As seen from above there was no increase in absorbance as one would expect. There is a decrease in absorbance for the enzyme plus pyroglutamine, which one might expect if the indigoidine being formed was precipitating out of solution. However Takahashi et al. saw an increase in absorbance when characterizing BPSA, an IndC homologue in vitro.
From this hypothesis it is noted that the oxidation domain in IndC most likely works on the glutamine while it is tethered to the PCP. If that is the case it would most likely fix the glutamine in a cis configuration, bringing together the residue amine group to act as an intramolecular nucleophile in order to finalize the cyclization of pyroglutamine. To test this new hypothesis, we are currently synthesizing a S-N-acetylcystamine (SNAC), which mimics the phosphopantathiene arm found on the PCP. The use of peptide SNAC to probe NRPS domain substrate specificity has been previously demonstrated .
We hope that by understanding the mechanism of glutamine cyclization and oxidation we can begin to create more robust IndC variants.
1. Hahn, M., & Stachelhaus, T. (2004). Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proceedings of the National Academy of Sciences of the United States of America, 101(44), 15585-15590.
2. Takahashi, H., Kumagai, T., Kitani, K., Mori, M., Matoba, Y., & Sugiyama, M. (2007). Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis. Journal of Biological Chemistry, 282(12), 9073-9081.
3. Marahiel, M. A., & Essen, L. O. (2009). Nonribosomal peptide synthetases: mechanistic and structural aspects of essential domains. Methods in enzymology, 458, 337-351.