1 INTRODUCTION
Due to remarkable advantages over
many small molecule drugs, such as lower toxicity and enhanced
selectivity, peptide drugs have been applied to a wide range of diseases
including cancer, bacillosis and immunological
rejection.1, 2Nowadays, peptide backbones
originating from microbial
biosynthesis become one of important sources for drug development.
However, L-amino acid as the common peptide unit is very vulnerable to
proteases and peptidases, which limits the medical application of
peptide compounds. Therefore, the introduction of D-amino acids is
regarded as an effective approach to enhancing the biostability and
bioavailability of peptide drugs, which can withstand the hydrolysis
activity of most endogenous enzymes and endow peptides stereochemical
properties.3 In general, the enzyme responsible for
epimerization always produces an equilibrating pool of L- and
D-antipodes or diastereomers, from
which the downstream enzyme exclusively selects D-configured peptide
chain for the following process.4
Uniquely, nocardicin thioesterase NocTE, which develops additional
epimerization activity and stereochemical selectivity, prefers D-Hpg to
L-Hpg in the C-terminal of products with high diastereomeric purity
(Figure 1A).5, 6 The bifunctional thioesterase
participates in the biosynthesis of β-lactam antibiotic nocardicin A as
an essential domain of the nonribosomal synthetase
NocB.7 Nocardicin A is active against various
gram-negative bacteria and possesses β-lactamase
resistance.5, 8, 9 During its biosynthesis,
nonribosomal peptide synthetases NocA and NocB assemble a precursor
peptide using nonproteinogenic D-(p-hydroxyphenyl)glycine (D-Hpg)
residues and some common proteinogenic residues
(L-Hpg-L-Arg-D-Hpg-L-β-lactam-D-Hpg)
(Figure 1B). The precursor peptide
contains a C-terminal core peptide and an
N-terminal follower peptide that has
been proven to determine substrate selectivity for NocTE in previous
studies.10 After the cleavage of N-terminal follower
peptide, the tripeptide nocardicin G is generated as the core structure
of nocardicin A. Nocardicin G contains two D-Hpg residues flanking a
β-lactam ring (D-Hpg-L-β-lactam-D-Hpg). The N-terminal D-Hpg is produced
by the typical epimerization domain, while the C-terminal D-Hpg
formation is catalyzed by bifunctional thioesterase NocTE.