Biosynthesis, Regulation and Export of Lasso Peptides - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 86

natural sequence, our group showed that the MccJ25 synthetase is able to tolerate
more dramatic changes and generate variants with insertions ([insA5-6], [insG22],
[insD22]) or deletions on the sequence ([ΔF10-V11], [ΔG12-G14]; Ducasse et al.
2012b ). These variants were either a lasso or a branched-cyclic topoisomer, further
demonstrating the versatility of the MccJ25 synthetase. Valuable insights into the
sequence requirement for each maturation step were provided by a unique in vitro
study, because MccJ25 maturation enzymes constitute the only available recombi-
nant system to date (Yan et al. 2012 ). This study used the ratio between the linear
and cyclized product to differentiate between the McjB and McjC activities. The
results indicate that McjB has a strict preference for Gly1 and relaxed specificity for
other residues, whereas the cyclization activity of McjC requires absolutely Glu8
and is influenced by residues in the tail region (e.g. Tyr19 or Tyr20). It remains to
determine the sequence requirement for the two steps catalyzed by McjC separately
(carboxylate adenylation and lactam formation).
In the case of capistruin, alanine scan of the core sequence revealed that the
maturation machinery absolutely requires Gly1 and Asp9, which are involved in the
macrolactam ring formation, and Arg11 located in the β-turn motif of the loop-and-
tail (Knappe et al. 2009 ). No detectable peptide masses attributable to G1A, G1C,
D9E, R11A and R11K substitutions could be found in the cell extracts. While the
substitution of Val12 and Ile13 separately to Ala abolished capistruin production,
single replacement to leucine at either position was accepted. Doubly mutated vari-
ants, R15A/F16A and F16A/F18A, but not the triple mutant R15A/F15A/F18A,
could be produced by the processing enzymes. Notably, R15A/F16A was produced
in vivo as two topoisomers (lasso and branched-cyclic forms).
Mutagenesis of caulosegnin I revealed new features of the maturation enzymes
(Hegemann et al. 2013a ). For the first time, replacement of Gly1 for Cys or Ala
resulted in detectable amounts of the corresponding lasso peptides, albeit with very
low yields. However, Phe1 was not tolerated. Truncation of the C-terminal tail had
effects on the maturation process, as variants ⊗G19, ⊗Q18-G19 and ⊗I17-G19
were produced with decreased efficiency. Single substitutions of residues in the
loop and the threaded tail region, including Pro12, Arg15 and Glu16 (the plug resi-
due) influenced the peptide production to various extents, which is independent of
the residue nature. In agreement with studies on MccJ25 and capistruin, change
of Glu8, which is involved in the isopeptide linkage, to Asp abolished completely
caulosegnin I production. Several single mutants of caulosegnin II and III were also
produced when their tail sequences were subjected to alanine scan for identifying
the plug residue.
The astexins processing system is remarkable because it allows substitution of
one ring-forming residue Asp9 to Glu in astexin-1 (Zimmermann et al. 2013 ). The
production level was very low and the D9E variant was likely to be in the branched-
cyclic form. On the contrary, the Gly1 to Cys substitution was not permitted. Single
substitutions by Ala of the loop-and-tail sequences revealed high overall tolerance
towards astexin-1 production, except for Tyr14 and Phe15. Shortening the size of
the tail by up to seven residues from the C-terminus was tolerated, whereas trunca-
tion of eight or nine residues had detrimental effects on the maturation process.