Biomedical Engineering Reference
In-Depth Information
Xanthomonins are recently discovered lasso peptides with the smallest ring size
(7 residues and 23 atoms; Hegemann et al.
2014
). The macrolactam ring is estab-
lished between Gly1 and the side-chain carboxylate of Glu7. Interestingly, a Glu is
also present at position 8, but it does not act as a ring-forming residue. Attempts to
change the ring size by constructing single or double substitutions failed, as E7A,
E7D, E7A/E8D and E7D/E8A completely abolished xanthomonin I production,
whereas E8A was produced with a good yield. Therefore, xanthomonins-processing
enzymes appear to be highly specific for forming a 23-atom ring. Moreover, the
exchange of Gly10 or Gly11 to any larger amino acid, such as Ala, Leu or Phe,
dramatically reduced the peptide production to a barely detectable level. Gly10 and
Gly11 are located immediately above the ring and their involvement in the matu-
ration process would be related to the intrinsic properties of a constrained seven-
residue ring.
Collectively, the above-cited studies lead to some general conclusions for the
specificity of lasso synthetases, although each lasso system has its own features.
Firstly, lasso-processing enzymes display promiscuity towards amino acid substitu-
tions on most positions of the core peptides. This is also reflected by the existence of
multi-precursor-encoded lasso gene clusters in nature. Secondly, they show a strin-
gent preference for native residues at position 1 and 7/8/9 that form the isopeptide
linkage. The proteolysis specificity (B protein function) is determined mainly by the
P1′ and P2 positions of the protease recognition site (nomenclature from Schechter
and Berger; Schechter and Berger
1967
). In contrast, the cyclization specificity (C
protein function) is conferred by the Asp or Glu residue involved in the macrolac-
tam ring formation and is influenced by some residues in the C-terminal tail region.
Thirdly, lasso synthetases are capable of producing both lasso and branched-cyclic
topoisomers, although it is likely that the branched-cyclic forms result from the
unthreading of unstable lasso peptides after their biosynthesis.
4.1.4
Maturation Reaction Model and Remaining Questions
The current working model of lasso peptide biosynthesis was put forth on the basis
of the in vitro characterization of MccJ25 maturation enzymes (Yan et al.
2012
;
Fig.
4.1
). The linear precursor peptide first undergoes a folding step in which the
C-terminal core peptide adopts a near-native conformation. The energy required for
this process is provided by ATP hydrolysis catalyzed by the B protein of the lasso
synthetase. Following leader peptide cleavage, the C protein activates the side-
chain carboxylate of Asp or Glu via an acyl-AMP intermediate and catalyzes the
macrolactam ring formation. This process is intriguing in view of the complexity
and number of peptide-protein, protein-protein and ligand (ATP)-protein interac-
tions involved. Understanding of the molecular mechanism of lasso synthetase is
one of the “hotspots” in lasso peptide research. Key aspects remain to be investigat-
ed, including the specificity determinants of lasso synthetases for cognate precursor
peptides, the precise mechanism of B proteins and B1/B2 complexes and structural
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