Biosynthesis, Regulation and Export of Lasso Peptides - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 89

The peptidase activity is specific towards the lasso topology, as AtxE2 cannot
cleave the branched-cyclic astexin-2. The kinetic parameters of AtxE2 were de-
termined using astexin-3 as substrate. The topology change of astexin-3 from the
lasso to the branched-cyclic form was followed by tryptophan fluorescence of the
peptide. AtxE2 exhibited a K m value of 131 µM, a k cat value of 0.38 s −1 and a k cat / K m
value of 2890 M −1 s −1 . AtxE2 belongs to the prolyl oligopeptidase family, a group
of serine proteases, and its active site was confirmed to be Ser257 by mutagenesis.
As for AtxE1 from the astexin-1 pathway, its ability to hydrolyze the lasso astexin-1
was proved in vivo. The functional role of such highly specific peptidases that rec-
ognize the interlocked lasso topology is intriguing. They catalyze the reverse reac-
tion of lasso peptide synthesis. Could it be a kind of self-protection strategy? Or as
Link and coworkers proposed, could it be a means to release a cargo from the lasso
peptide? These questions await deeper understanding of the ecological roles of the
respective lasso peptides.

4.2 Regulation of Lasso Peptide Production

Currently, little information is available for regulation of lasso peptide production.
It is conceivable that, like other secondary metabolites from bacteria, the production
of lasso peptides is subjected to complex regulation by environmental and genetic
factors. Most of the lately studied proteobacteria produced no or very weak amount
of lasso peptides under laboratory conditions, which highlights the importance of
understanding their regulation mechanisms.
First insights were provided for MccJ25 (Chiuchiolo et al. 2001 ). MccJ25 pro-
duction was shown to be induced under iron-deficient conditions (Salomon and
Farias 1994 ) and to increase dramatically at the stationary phase in both LB and
M63 minimal media. The growth phase-dependent behaviour was studied in detail
using a translational fusion of mcjA, coding for MccJ25 precursor peptide, and the
reporter gene lacZ . Reduction of the growth rate upon transition from exponential
to stationary phase triggered the expression of mcjA . It was demonstrated that this
induction is due to carbon and phosphate limitation, and is independent of nitro-
gen depletion, pH change and cell density. At the molecular level, mcjA expres-
sion is positively regulated by a complex network of global regulators, including
at least the leucine-responsive regulatory protein (Lrp), the integration host factor
(IHF) and the highly phosphorylated guanosine nucleotide (p)ppGpp. Inspection
of mcjA promoter region identified putative Lrp and IHF binding sites (Craig and
Nash 1984 ; Rex et al. 1991 ; Chiuchiolo et al. 2001 ), consistent with the experimen-
tal data. It was suggested that the (p)ppGpp is the main positive effector of mcjA
expression given that it positively controls the expression of genes encoding Lrp
and IHF (Aviv et al. 1994 ; Landgraf et al. 1996 ; Chiuchiolo et al. 2001 ). This effect
of (p)ppGpp is reminiscent of the regulation of antibiotic production in Streptomy-
ces (Bibb 2005 ), in agreement with the antibacterial role of MccJ25. It was shown
that induction of mcjA by (p)ppGpp accumulation is dependent on the spoT gene