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posted on 27.02.2018, 01:31 authored by Max CryleMax Cryle

Accepted manuscript:

An enhanced chemo-enzymatic method for loading substrates onto carrier protein domains

Tiia Kittilä and Max J. Cryle

Biochemistry and Cell Biology

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.