Biomedical Engineering Reference
In-Depth Information
basis for lasso synthetases. However, at present, the in-depth study of lasso syn-
thetase enzymology is hampered by the difficulty in obtaining soluble and pure
maturation enzymes. Huge amounts of efforts have been made in our laboratory to
improve McjB/McjC production. It turned out that maltose-binding protein (MBP)
fused to the N-terminus of McjB led to an active protein produced in high yield. In
the case of McjC, coexpression with chaperones was the best strategy. However, the
removal of the copurified chaperones from McjC is a challenge. It is not uncommon
that enzymes from other lasso peptide pathways are insoluble (Marahiel MA, per-
sonal communications); this is probably related to the intrinsic properties of these
maturation enzymes. A systematic approach will be needed to screen available lasso
synthetases in diverse genomes for targets suitable for recombinant production.
How lasso synthetases function in vivo is an important unanswered question. It
remains to demonstrate the relevance of the complex formation in vivo, to localize
the synthetase in the cell and hence where the maturation takes place, and to iden-
tify additional partners that might be involved in the maturation process (e.g. the
transporter). Moreover, it is unknown if the lasso peptide synthesis and transport
are tightly coupled as in the case of lantibiotics (Alkhatib et al.
2012
). It was shown
that the MccJ25-synthesizing activity was found in the membrane fraction of cell
extracts (Clarke and Campopiano
2007
), suggesting that the synthetase is probably
located at the membrane. Pull-down assays and in cell imaging techniques might
help addressing these questions.
4.1.5
Accessory Enzymes
The genetic system of MccJ25 and capistruin, which consists of four
ABCD
genes
responsible for the peptide biosynthesis and transport, has been considered for a
long time the archetype of lasso clusters. With the expansion of available bacte-
rial genome sequences, genome mining studies revealed other genes encoded in
the immediate genomic context of the lasso biosynthetic genes and hinted their
involvement in the modification or regulation of lasso peptides (Maksimov et al.
2012
; Hegemann et al.
2013b
). Frequent occurrences are genes encoding sulfo-
transferases, protein kinases, methyltransferases, acetyltransferases, peptidases and
regulators. However, the majority of them have not been experimentally validated.
Remarkably, a particular gene organization is conserved in proteobacteria, which is
obligatorily composed of two genes encoding a prolyl oligopeptidase and a TonB-
dependent receptor, respectively, in addition to the
ABC
biosynthetic genes. They
are often accompanied by a GntR-type regulator and a pair of FecI/FecR homo-
logues (sigma factor and anti-sigma factor).
The peptidases encoded by the astexins clusters in
Asticcacaulis excentricus
have been functionally characterized recently (Maksimov and Link
2013
), adding a
new validated member to lasso modification enzymes. In vitro assays showed that
AtxE2 from the astexin-2 and astexin-3 pathway hydrolyzes the isopeptidic bond
of these lasso peptides to yield the linear form, thus functioning as an isopeptidase.
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