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.