Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines

Abstract

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.

Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold

“…In another study, cyclo(L-Phe-L-Pro) produced by Vibrio vulnificus was found to activate the quorum sensing on an E. coli biosensor strain by modulating the expression of the gene encoding OmpU protein [231]. Finally, cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) produced by Lactobacillus reuteri, were reported to interfere with quorum sensing in the Gram-positive S. Aureus [41,221,227,[232][233][234].…”

Modifications of diketopiperazines assembled by cyclodipeptide synthases with cytochrome P450 enzymes

“…Very recently, two groups published the structure of the same P 450 from Streptomyces sp. NRRL F-5053, termed NzeB and Nas F5053 , respectively (Shende et al 2020 ; Sun et al 2020 ). It was proposed that the dimerisation of cWP catalysed by this enzyme would also be via a radical-mediated mechanism (Fig.…”

“…8 ) (Shende et al 2020 ). In contrast to CYP121, this dimerase only forms one radical at nitrogen N1 (Sun et al 2020 ) or N12 (Shende et al 2020 ) after abstraction of one hydrogen by compound I (intermediate 1). In the mechanism proposed by Shende et al ( 2020 ), the resulting radical then shifts to C3 after cyclisation between N12 and C2 (intermediate 2).…”

“…Sequence alignments of NascB, NzeB (synonym Nas F5053 ) and Nas S1868 (synonym AspB) revealed four critical amino acid residues in these P 450 s controlling the regio- and stereoselectivity. Mutation on the key residues at positions 65, 86, 284 and 288 led to the alteration of the regio- and stereospecificity of these enzymes (Sun et al 2020 ).…”

Diketopiperazine Gels: New Horizons from the Self-Assembly of Cyclic Dipeptides

“…Interestingly, a cytochrome P450 has been identified that can catalyze both the CDP dimerization and cyclization towards bioactive CDP-derived natural products with interesting bioactivities ( Figure 2 ) [ 96 ]. Mutagenesis of other cytochrome P450s gave access to catalysts for CDP synthesis with good regio- and stereo-specificity, and chemical versatility [ 97 ]. Whole-cell biocatalysts were used to attain heterodimeric CDPs, with Mycobacterium smegmatis demonstrating to be a more robust and efficient organism, than the commonly used Escherichia coli or Streptomyces systems [ 98 ].…”