Team:Queens Canada/Description

Team:Queens Canada





Investigating IndC Robustness




In 2013, Heidelberg iGEM introduced indigoidine tagging of nonribosomal peptides (NRPs) to the iGEM community. It was noted however that there was no paper published on the ability of indigoidine to tag NRPs, nor were there any nuclear magnetic resonance (NMR) or mass spectrometry (MS) data present to demonstrate its ability to tag NRPS.

The Heidelberg 2013 team mentioned that they experienced trouble tagging bulky peptides such as tryptophan. Therefore, one possibility we wanted to explore was steric hindrance of the oxidation domain found in the IndC module incorporating indigoidine.

One other reason behind further investigating the oxidation domain in IndC, the NRPS module responsible for indigoidine synthesis, was because the exact mechanism of indigoidine formation was unknown. It has been debated whether the oxidation domain acts on the cyclized final compound as seen in other FMN-dependent oxidation domains found within NRPS systems, or whether it would oxidize the glutamine attached to the PCP.



Our first hypothesis was the former mentioned above, that the oxidation domain acts on the cyclized product after it is released by the thioesterase (TE) domain. This hypothesis was derived from the fact that cyclized glutamine was observed as the final amino acid in the molecule Padanamide B.




This leads us to believe that the TE domain would cyclize glutamine, which would then be oxidized by the oxidation domain in order to spontaneously combine into indigoidine.

The precursor was purchased from Astatech, and it was dissolved in water. The pyroglutamine had a blue colour in solution with absorbance similar to indigoidine at 600nm. However, the pyroglutamine was soluble in water and insoluble in DMSO, opposite of what indigoidine was found to be soluble in in literature [2].

NMR was carried out in order to determine if the compound we ordered was indeed pyroglutamine. The compound was dissolved in deuterated ethanol, and the NMR spectrum is pictured below.





Despite the solution containing a blue colour, no traces of indigoidine were observed.

The oxidation domain was ordered from IDT and cloned into a pET-16b vector containing a 10x C-terminal His tag. It was purified via nickel affinity chromatography. The final concentration was 12.2 mg/ml of protein. The purified oxidation domain contained a yellow colour demonstrating that it was co-purifying with bound FMN.

An enzymatic assay was carried out by adding pyroglutamine and our enzyme to HEPES buffer (pH 7.2) in a plastic cuvette and scanning for changes in absorbance at 600nm every minute for ten minutes total.





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 [3].

We hope that by understanding the mechanism of glutamine cyclization and oxidation we can begin to create more robust IndC variants.



References



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.