Structure and Function of NzeB, a Versatile C–C and C–N Bond-Forming Diketopiperazine Dimerase

Abstract

The dimeric diketopiperazine (DKPs) alkaloids are a diverse family of natural products (NPs) whose unique structural architectures and biological activities have inspired the development of new synthetic methodology to access these molecules. However, catalystcontrolled methods that enable the selective formation of constitutional and stereoisomeric dimers from a single monomer are lacking. To resolve this long-standing synthetic challenge, we sought to characterize the biosynthetic enzymes that assemble these NPs for application in biocatalytic syntheses. Genome mining enabled identification of the cytochrome P450, NzeB (derived from Streptomyces sp. NRRL F-5053), which catalyzes both intermolecular carbon-carbon (C-C) and carbon-nitrogen (C-N) bond formation, generating all currently known DKP dimer scaffolds isolated from bacterial sources. To identify the molecular basis for the flexible site-, stereo-, and chemoselectivity of NzeB, we obtained high-resolution crystal structures (1.5Å) of the protein in complex with native and non-native substrates. This, to our knowledge, represents the first crystal structure of an oxidase catalyzing direct, intermolecular C-H amination. Site-directed mutagenesis was employed to assess the role individual active site residues play in guiding selective DKP dimerization. Finally, computational approaches were employed to evaluate plausible mechanisms regarding NzeB function and its ability to catalyze both CC and C-N bond formation. These results provide a structural and computational rationale for the catalytic versatility of NzeB, as well as new insights into variables that control selectivity of CYP450 diketopiperazine dimerases. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

Modifications of diketopiperazines assembled by cyclodipeptide synthases with cytochrome P450 enzymes

“…CMB-MQ030 (Tian et al 2018 ) and NzeB from Streptomyces sp. NRRL F-5053 (Shende et al 2020 ) are cWP dimerases for C3-C5´ coupling with naseazine C as the main product. Two two-gene operons from Saccharopolyspora antimicrobica DSM 45119 are responsible for the formation of tetratryptomycins with four tryptophanyl units.…”

“…The aforementioned NzeB also catalyses the N1-C6´dimerisation of two cWP molecules, resulting in the formation of aspergilazine A as minor side product (Shende et al 2020 ). Aspergilazine A was identified as the main product of a two-gene BGC from Streptomyces sp.…”

“…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.…”

“…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).…”

“…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).…”