Promiscuous Enzymes Cooperate at the Substrate Level En Route to Lactazole A

Enzymes
involved in the biosynthesis of ribosomally synthesized
and post-translationally modified peptides (RiPPs) often have relaxed
specificity profiles and are able to modify diverse substrates. When
several such enzymes act together during precursor peptide maturation,
a multitude of products can form, yet usually the biosynthesis converges
on a single natural product. For the most part, the mechanisms controlling
the integrity of RiPP assembly remain elusive. Here, we investigate
the biosynthesis of lactazole A, a model thiopeptide produced by five
promiscuous enzymes from a ribosomal precursor peptide. Using our
in vitro thiopeptide production (FIT-Laz) system, we determine the
order of biosynthetic events at the individual modification level
and supplement this study with substrate scope analysis for participating
enzymes. Our results reveal an unusual but well-defined assembly process
where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation
events are intertwined due to minimal substrate recognition requirements
characteristic of every lactazole enzyme. Additionally, each enzyme
plays a role in directing LazBF-mediated dehydroalanine formation,
which emerges as the central theme of the assembly process. Cyclodehydratase
LazDE discriminates a single serine residue for azoline formation,
leaving the remaining five as potential dehydratase substrates. Pyridine
synthase LazC exerts kinetic control over LazBF to prevent the formation
of overdehydrated thiopeptides, whereas the coupling of dehydrogenation
to dehydroalanine installation impedes generation of underdehydrated
products. Altogether, our results indicate that substrate-level cooperation
between the biosynthetic enzymes maintains the integrity of lactazole
assembly. This work advances our understanding of RiPP biosynthesis
processes and facilitates thiopeptide bioengineering.