An enhanced chemoenzymatic method for loading substrates onto carrier protein domains

Abstract

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 to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto 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 chemoenzymatic loading method that combines 2 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 2 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.

Using delayed decoupling to attenuate residual signals in editing filters

“…In the current work, we focus on the carrier protein PCP1 from yersiniabactin synthetase, which natively harbors a cysteine substrate (Gehring et al, 1998). In order to study interactions between PCP1 and the phosphopantetheine group and its attached cysteine substrate, we have chemoenzymatically (Kittilä and Cryle, 2018;Worthington and Burkart, 2006) attached the unlabeled PP group and substrate to PCP1 enriched in 15 N and 13 C isotopes (Fig. 1).…”

“…Yousang Hwang, Johns Hopkins chemistry core facility) followed methods described by (Worthington and Burkart, 2006) and improved by (Kittilä and Cryle, 2018) with the following adaptations. Apo PCP1 was thawed and buffer exchanged into one-pot reaction buffer: 100 mM Tris pH 7.55 at 22 °C, 10 mM MgCl2, 100 mM NaCl, and 2.5 mM DTT.…”

“…Apo PCP1 was thawed and buffer exchanged into one-pot reaction buffer: 100 mM Tris pH 7.55 at 22 °C, 10 mM MgCl2, 100 mM NaCl, and 2.5 mM DTT. To a 10-mL reaction at 25 °C, apo PCP1 (118 μM), Cys-NH-pantetheine analogue (509.4 μM), ATP (1 mM), PanK (2.5 μM), PPAT (2.5 μM), DPCK (2.5 μM), and Sfp R4-4 (5.0 μM) were mixed together following sequential addition-incubation steps as in (Kittilä and Cryle, 2018). Preparation of PanK, PPAT, and DPCK was performed as described (Goodrich et al, 2017).…”

Recent progress in enzymatic protein labelling techniques and their applications

“…292 Together with an uncharacterized carboxypeptidase, the C-terminus of α-tubulin is subjected to a post-translational process where a tyrosine residue can be reversibly added and removed, which is important in regulating microtubule function. 293 TTL suppression has been reported to be associated with progression of neuroblastoma, 294 and even in normal cells, TTL has been shown to play a vital role in controlling neuronal organization. 295…”

PKS–NRPS Enzymology and Structural Biology: Considerations in Protein Production

“…The promiscuous Sfp from Bacillus subtilis has been the most widely employed because of its high catalytic activity and relaxed substrate specificity (Lambalot, Gehring, Flugel, Zuber, LaCelle, Marahiel et al, 1996), but Streptomyces verticillus Svp has proven to be equally versatile (Sánchez, Du, Edwards, Toney & Shen, 2001). Additionally, an Sfp mutant (R4-4 Sfp) will load the simpler and more synthetically accessible 3-dephospho-CoA analogues (Zou & Yin, 2009; Kittilä & Cryle, 2017).…”