Accepted_Manuscript_BCB_Kittila_2017.pdf
Max Cryle
10.4225/03/5a94b50928032
https://bridges.monash.edu/articles/preprint/Accepted_Manuscript_BCB_Kittila_2017_pdf/5926615
<p>Accepted manuscript:</p><p>
</p><p><b>An
enhanced chemo-enzymatic method for loading substrates onto carrier protein
domains</b><b></b></p>
<p>Tiia Kittilä
and Max J. Cryle</p>Biochemistry and Cell Biology<p></p>
<p>Non-ribosomal peptide synthetase
(NRPS) machineries produce many medically relevant peptides that cannot be easily
accessed by chemical synthesis. Thus, understanding NRPS mechanism is of
crucial importance to allow efficient redesign of these machineries in order to
produce new compounds. During NRPS-mediated synthesis, substrates are
covalently attached to PCPs, and studies of NRPSs are impeded by difficulties
in producing PCPs loaded with substrates. Different approaches to load
substrates on to PCP domains have been described, but all suffer from
difficulties in either the complexity of chemical synthesis or low enzymatic efficiency.
Here, we describe an enhanced chemo-enzymatic loading method that combines two
approaches into a single, highly efficient one-pot loading reaction. First, d-pantetheine and ATP are converted into
dephospho-coenzyme A via the actions of two enzymes from coenzyme A (CoA)
biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline
phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which
is inhibited by phosphoadenylates. This route does not depend on activity of
the commonly problematic dephospho-CoA kinase, and therefore offers an improved
method for substrate loading onto PCP domains.</p><p><br></p><p><a href="https://doi.org/10.1139/bcb-2017-0275">https://doi.org/10.1139/bcb-2017-0275</a></p>
2018-02-27 01:31:51
carrier proteins Cp
Post-translational Modifications of Proteins
non-ribosomal peptide synthetases
phosphopantetheinyl transferases
Coenzyme APantetheine
Biochemistry and Cell Biology not elsewhere classified