4 research outputs found
Adenylation and <i>S</i>āMethylation of Cysteine by the Bifunctional Enzyme TioN in Thiocoraline Biosynthesis
The antitumor agent thiocoraline
is a nonribosomally biosynthesized
bisintercalator natural product, which contains in its peptidic backbone
two <i>S</i>-methylated l-cysteine residues. <i>S</i>-Methylation occurs very rarely in nature, and is observed
extremely rarely in nonribosomal peptide scaffolds. We have proposed
that during thiocoraline biosynthesis, TioN, a stand-alone adenylation
domain interrupted by the <i>S</i>-adenosyl-l-methionine
binding region of a methyltransferase enzyme, is capable of performing
two functions: the adenylation and <i>S</i>-methylation
of l-cysteine. Herein, by preparation of knockouts of TioN
and its MbtH-like protein partner TioT, we confirmed their role in
thiocoraline biosynthesis. We also co-expressed recombinant TioN and
TioT and biochemically investigated three potential pathways involving
activation, methylation, and loading of l-cysteine onto the
TioN partner thiolation domain, TioSĀ(T<sub>4</sub>). The valuable
insights gained into the pathway(s) followed for the production of <i>S</i>-Me-l-Cys-<i>S</i>-TioSĀ(T<sub>4</sub>) will serve as a guide for the development of novel engineered interrupted
adenylation enzymes for combinatorial biosynthesis
Activation and Loading of the Starter Unit during Thiocoraline Biosynthesis
The
initiation of the nonribosomal peptide synthetase (NRPS) assembly
of the bisintercalator natural product thiocoraline involves key enzymatic
steps for AMP activation and carrier protein loading of the starter
unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with
protein sequence homology analysis originally led us to propose that
TioJ could be responsible for the AMP activation step, whereas TioO
could act as the thiolation (T) domain, facilitating the transfer
of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement
of TioJ in thiocoraline biosynthesis by <i>tioJ</i> knockout
and <i>in vitro</i> activation of 3HQA studies. However,
we demonstrated that TioJ-activated 3HQA is not loaded onto the T
domain TioO, as originally believed, but instead onto a fatty acid
synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located
outside of the thiocoraline gene cluster. We showed a strong interaction
between TioJ and FabC. By generating TioJ point mutants mimicking
the active site of highly homologous enzymes activating different
molecules, we showed that the identity of the substrate activated
by adenylation domains such as TioJ is not determined by only the
active site residues that directly interact with the substrate. The
insights gained from these enzymatic transformations are valuable
in the efforts toward deciphering the complete biosynthetic pathway
of thiocoraline and bisintercalators in general