Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Abstract

18Enzymes involved in ribosomally synthesized and post-translationally modified peptide 19 (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse 20 substrates. When several such enzymes act together during precursor peptide maturation, 21 a multitude of products can form, and yet usually, the biosynthesis converges on a single 22 natural product. For the most part, the mechanisms controlling the integrity of RiPP 23 assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model 24 thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. 25Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of 26 biosynthetic events at the individual modification level, and supplement this study with 27 substrate scope analysis for participating enzymes. Combined, our results reveal a 28 dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined 29 enzymatic action, and the overall substrate-level cooperation between the enzymes. This 30 work advances our understanding of RiPP biosynthesis processes and facilitates 31 thiopeptide bioengineering. 32 3 Main text 33 Ribosomally synthesized and post-translationally modified peptides (RiPPs) are 34 structurally and functionally diverse natural products united by a common biosynthetic 35 logic. 1 Usually during RiPP maturation, biosynthetic enzymes utilize the N-terminal 36 sequence of a ribosomally produced precursor peptide as a recognition motif (leader 37 peptide; LP) and install post-translational modifications (PTMs) in the C-terminal section of 38 the same substrate (core peptide; CP). This mode of action leads to relaxed substrate 39 requirements around the modification sites, which is often exemplified by one RiPP 40 enzyme introducing multiple PTMs in a single substrate. In one extreme case, a single 41 enzyme epimerizes 18 out of 49 amino acids in polytheonamide A precursor peptide 42 during its biosynthesis. 2,3 Unique enzymology of RiPP biosynthetic enzymes has come 43 under intense scrutiny in the recent years, which explained observed substrate specificities 44 in many cases. 4,5,14,15,6-13 During biosynthesis of complex RiPPs, when multiple enzymes 45 capable of differentially modifying their substrate act together, a multitude of products can 46 often form, and yet usually the biosynthetic pathway manages to produce a single natural 47

Introduction to Thiopeptides: Biological Activity, Biosynthesis, and Strategies for Functional Reprogramming

“…Here, efficient Ser dehydration is contingent on the action of PbtH, a C-terminal ''amidase'' enzyme (vide infra). In contrast, lactazole maturation features a divergent sequence of events (Vinogradov et al, 2020a). Here, Ser/Cys cyclodehydration, azoline dehydrogenation, and Ser dehydration steps are intertwined, which results in a convoluted but overall well-defined biosynthetic order.…”

“…The assembly process can be roughly divided into pre-and post-macrocyclization stages. Current understanding regarding the order of events during the pre-macrocyclization stage primarily comes from the studies on in vitro assembly of thiomuracin and lactazole core macrocycles (Figure 3B) (Hudson et al, 2015;Vinogradov et al, 2020a;Zhang et al, 2016b), as well as in vivo studies of micrococcin P1 (Bennallack et al, 2016;Bewley et al, 2016) and GE2270A biosynthesis (Tocchetti et al, 2013).…”

“…A recent analysis of azole formation in sulfomycin (Figure S1), a berninamycin-like compound, revealed that two dehydrogenases form a heterocomplex capable of oxidizing oxazolines, although enzymatic activities of the individual proteins were not deconvoluted (Du et al, 2020). Slow oxazoline dehydrogenation by LazF is accelerated when the substrate is flanked by a dehydroalanine (Dha) residue, likely because conjugation between Dha and oxazoline p systems facilitates the reaction (Vinogradov et al, 2020a).…”

Site-specific nonenzymatic peptide S/O–glutamylation reveals the extent of substrate promiscuity in glutamate elimination domains

“…LazF is unique in the fusion of the ED to an FMN dehydrogenase, utilized for azoline to azole conversion during lactazole assembly. 31 The enzymes were heterologously expressed (S.I. 2.1) in Escherichia coli as soluble proteins following the established procedures.…”

LimF is a versatile prenyltransferase catalyzing histidine-C-geranylation on diverse nonnatural substrates

“…As RiPP enzymes are often affected by the local sequence environment nearby the modification site, [52][53][54][55][56] we commenced with mutagenesis of the residues adjacent to the geranylation site. We expressed thirty-six teLimE2 mutants, in which the His-1 and His+1 positions are replaced with 19 proteinogenic amino acids (expect Cys), in the FIT-LimF system, and the reaction outcomes were analyzed by LC-MS (Fig.…”