Functional Characterization and Crystal Structure of the Bifunctional Thioesterase Catalyzing Epimerization and Cyclization in Skyllamycin Biosynthesis

Abstract

The D-amino acid residues are hallmark building blocks of nonribosomal peptides. Here, we report the bifunctional thioesterase domain (TE domain) Skyxy-TE that catalyzes both epimerization and cyclization in skyllamycin biosynthesis. Skyxy-TE specifically catalyzes the epimerization of the C-terminal L-amino acid residue of the linear substrate, then catalyzes regioselective intramolecular cyclization. The crystal structure of Skyxy-TE was solved at 2.25 Å and site-directed mutagenesis was performed, revealing key residues involved in the epimerization and cyclization. This study expands the understanding of the versatile TE domains and facilitates chemoenzymatic synthesis or combinatorial biosynthesis in the future.

Epoxinnamide: An Epoxy Cinnamoyl-Containing Nonribosomal Peptide from an Intertidal Mudflat-Derived Streptomyces sp.

“…For detailed comparative analysis, the structures of these thioesterase domains were predicted by Phyre2 [ 39 ]. These two thioesterase domains showed a 60% of sequence identity to Skyxy-TE, which catalyzes both epimerization and macrocyclization in biosynthesis of skyllamycin [ 40 ]. Several amino acid residues exist (Pro31, Ala32, Trp96, Ser97, Leu98, Asp124, Gln125, Pro139, Phe202, and His254) that play important roles in the function of Skyxy-TE ( Figure S18 ).…”

“…Gln125 was replaced with Glu125, which is structurally analogous to Gln125. The side-chain amide group of Gln125 forms hydrogen bonds with adjacent main-chain amide oxygens in Skyxy-TE [ 40 ]. Since glutamic acid has a similar size to glutamine and also has a hydrogen atom capable of hydrogen bonding, we could predict that Epc-TE would still have both epimerizing and cyclizing functions, analogously to Skyxy-TE ( Figure S19 ).…”

“…Through these observations, we could conclude that Glx125 (Gln or Glu) is an important residue in the epimerization function. Thus, it would be possible to commonly identify the homologous active sites of the bifunctional thioesterase domains through amino acid sequence analysis and protein structure predictions of Epc-TE, Dml-TE, and Atr-TE [ 13 , 21 , 39 , 40 ].…”

Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism

“…The catalytic role of the active site histidine residue can also be seen in the epimerization activity of NocTE and Skyxy-TE. 10,11 Since the E domain epimerization is a reversible process as shown in Fig. 1, both catalytic residues must function as both acid and base with water in the active site assisting in proton shuttling between the residues to re-establish their roles in the epimerization for continuous activity.…”

“…9 More recently, crystal structures of two thioesterases, NocTE and Skyxy-TE, revealed the key residues involved in their unusual ability to also catalyze an epimerization reaction to include d -amino acids into their linear and cyclic peptides, specifically in norcardicin and skyllamycin biosynthesis, respectively. 10,11 However, the majority of NRPS-incorporated d -amino acids are converted from l -amino acids subsequent to covalent loading on PCPs through the use of auxiliary epimerization (E) domains. Unlike amino acid racemases, which utilize the PLP cofactor, E domains catalyze epimerization without the use of cofactors.…”