Synthetic biology of non-ribosomal cyclopeptide
Toshiyuki WAKIMOTO
Faculty of Pharmaceutical Sciences, Hokkaido University
Research objective
The macrocyclization of medium-sized molecules is considered one of the most challenging
reactions to achieve in organic synthesis. It is difficult to bring both ends of the linear substrate
close together, and in the case of peptides, epimerization of the C-terminal amino acid can leadto the formation of byproducts. In the presence of multiple reactive functional groups on thesubstrate, protecting groups are necessary to achieve the desired regioselectivity. Furthermore,high-dilution conditions are essential owing to the competition between the desired
intramolecular reactions and undesired intermolecular reactions. This can pose a serious
problem for large-scale synthesis processes. Considering natural products, several compoundswith macrocyclic skeletons have been biosynthesized efficiently by biosynthetic enzymes. By
discovering naturally occurring cyclases and attempting to modify their functions using
enzyme engineering and directed evolution, we could develop cyclases with diverse substratespecificities that can be employed for producing versatile macrocyclic medium-sizedmolecules.
In the present study, we modified penicillin-binding protein-type thioesterase (PBP-typeTE), a novel cyclase originally discovered by our group from an actinomycete, to develop abiocatalyst that can easily synthesize macrocyclic skeletons and realize a biosynthetic platform
for producing a variety of cyclic peptides. Given that macrocyclization of peptides andpolyketides can confer advantageous physical properties, such as fixation of the active
conformation and resistance to digestive enzymes, compared with linear structures, this study
provides a fundamental technology for potentially modifying medium-sized drug leads.
Methods
We discovered a new peptide cyclase, SurE, involved in the biosynthesis of surugamide, a
non-ribosomal cyclic peptide produced by actinomycetes, and elucidated its substrateselectivity and crystal structure. SurE is a 50 kDa "stand-alone" enzyme independent of nonribosomal peptide synthetase (NRPS), whereas cyclases of the NRPS pathway are typically
fused to the C-terminus of a giant enzyme of several mega Da (MDa). Owing to its homology
to PBP, we classified SurE as a novel peptide cyclase family, PBP-type TE. SurE can be easily
prepared as a recombinant enzyme and has high potential as a biocatalyst, given its wide
substrate selectivity and catalytic efficiency, comparable with that of commercially available
peptide cyclization biocatalysts.
PBP-type TE-mediated non-ribosomal peptide biosynthesis is a universal biosynthetic

mechanism distributed in more than 10% of actinomycetes and is responsible for thebiosynthesis of cyclic peptides of various structures. These PBP-type TE homologs exhibit
high amino acid sequence homology with each other but display completely different substrateselectivities from that of SurE. Herein, we elucidated the molecular basis of the substrate
recognition mechanism by biochemically and structurally comparing multiple PBP-type TEs
that exhibit high sequence homology but distinct substrate selectivities. Accordingly, we canobtain fundamental knowledge for freely controlling selectivity to design improved
biocatalysts with extended substrate and reaction selectivity, as well as develop novel efficientsynthetic methods for cyclic peptides.
Results
More than 700 candidate genes for PBP-type TE are available in the public genome
databases. The identified genes were subjected to network analysis based on amino acidsequence similarity and found to be classified into clusters of approximately 40 types
according to substrate selectivity. This analysis enabled the efficient identification of novel
homologous enzymes. Therefore, we selected 10 homologous enzymes representing eachclade and attempted to clone and express them heterologously. Plasmids for recombinant
protein expression were prepared, and the recombinant enzymes expressed in Escherichia coli
were purified by affinity chromatography with Ni resin. Subsequently, 8 of the 10 homologous
enzymes were obtained as insoluble enzymes. Two proteins were identified as soluble
enzymes (Nsm16 and SurE14988). Furthermore, PenA, which is responsible for thecyclization of the cyclic pentapeptide pentaminomycin, was cloned from Streptomyces cacaoi
NBRC 12748, and heterologous expression was attempted in E. coli. Given that PenA provides
a cyclic peptide with a smaller ring size than SurE, we performed an in-depth assessment of
its substrate specificity. Based on the observed results, although PenA is unable to catalyze thecyclization of surugamide B, it was found to specialize in short-chain substrates (Fig. 1). SurE
could cyclize peptides of 5–11 amino acid residues; for tetrapeptide generation, dimerization
also occurred in addition to cyclization. In contrast, PenA cyclized peptides with 4–5 aminoacid residues and selectively catalyzed the cyclization of tetrapeptides without dimerization(Fig. 1). The PenA model structure revealed a loop structure that may be responsible for its
unique selectivity, confirming that PenA could cyclize shorter peptide substrates than SurE.1
To structurally elucidate the mechanism underlying this unique selectivity, we generated amodel structure of PenA using the SurE structure (PDB:6KSU) as a template and compared itwith the model structure of SurE. The results revealed that a portion of the loop constituting
the C-terminal lipocalin domain of PenA was markedly elongated, protruding toward thesubstrate-binding pocket (Fig. 1). These loops narrow the substrate pocket volume, which islikely to be more suitable for short peptides.
DsaJ is a PBP-type TE that is responsible for the biosynthesis of cyclic hexapeptide

desotamides. While common PBP-type TEs selectively cyclize substrates with a D-amino acidresidue at the C-terminal end, DsaJ cyclizes substrates with an achiral Gly residue at the Cterminal end. To gain insight into the structural basis for this unique selectivity, a model DsaJ
structure was constructed using AlphaFold 2 and compared with that of SurE, suggesting thatthe hydrophobic pocket surrounding the catalytic serine residue is critical for the C-terminal
D-amino acid selectivity of the SurE substrate. In the model DsaJ structure, the residues
constituting the hydrophobic pocket are replaced with bulky residues. The unique substrateselectivity of DsaJ could be attributed to the pocket structure, and the residues comprising thepocket were replaced by bulky residues in SurE. Accordingly, the mutant enzyme lost its
activity against the original substrate (C-terminal residue D-Leu) but was able to cyclize a new
substrate with a C-terminal Gly residue. These results indicated that the hydrophobic pocket
is pivotal for recognizing the C-terminal residue of the substrate, and the selectivity of the Cterminal residues of SurE and DsaJ was completely switched.
Conclusion
Focusing on the substrate specificity of naturally occurring PBP-type TE, we attempted toperform a detailed functional analysis and functional modification by enzyme engineering,
successfully developing new cyclases compatible with various substrates. Currently, we aredeveloping more efficient methods for synthesizing cyclic peptides using these cyclases.

Fig. 1. Functional analysis of PenA
References
1) Matsuda, K., Fujita, K., and Wakimoto, T. (2021) PenA, a penicillin-binding protein-type
thioesterase specialized for small peptide cyclization. J. Ind. Microbiol. Biotechnol. 48:
kuab023.