Total Enzyme Syntheses of Napyradiomycins A1 and B1

Abstract

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon−carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.

Bromination of alkyl quinolones by Microbulbifer sp. HZ11, a marine Gammaproteobacterium, modulates their antibacterial activity

“…Vanadium-dependent haloperoxidases require vanadate and use hydrogen peroxide for halide oxidation, yielding hypohalite (HOX) intermediates chemically equivalent to an electrophilic halenium ion 'X + ' for halogenation of electron rich substrates (Butler, 1998;Butler and Carter-Franklin, 2004;Winter and Moore, 2009). Although initially described as enzymes producing hypohalous acids as diffusible halogenating agent, thereby lacking substrate specificity or regioselectivity (Itoh et al, 1988;van Pée and Patallo, 2006), recently several biosynthetic Streptomyces V-HPOs, among them NapH1, have been identified to catalyse stereoselective halogenation reactions, suggesting an enzyme-bound halenium ion rather than free hypohalous acid as the halogenating agent (Winter and Moore, 2009;McKinnie et al, 2018). These selective enzymes participate in complex biosynthetic pathways Carter-Franklin and Butler, 2004;Winter et al, 2007;Bernhardt et al, 2011;McKinnie et al, 2018).…”

“…Although initially described as enzymes producing hypohalous acids as diffusible halogenating agent, thereby lacking substrate specificity or regioselectivity (Itoh et al, 1988;van Pée and Patallo, 2006), recently several biosynthetic Streptomyces V-HPOs, among them NapH1, have been identified to catalyse stereoselective halogenation reactions, suggesting an enzyme-bound halenium ion rather than free hypohalous acid as the halogenating agent (Winter and Moore, 2009;McKinnie et al, 2018). These selective enzymes participate in complex biosynthetic pathways Carter-Franklin and Butler, 2004;Winter et al, 2007;Bernhardt et al, 2011;McKinnie et al, 2018).…”

New Napyradiomycin Analogues from Streptomyces sp. Strain CA-271078

“…CNQ-525, and its analysis established the link between the presence of three vanadium-dependent haloperoxidases (VHPOs), (NapH1, NapH3, and NapH4) and a chloronium-induced meroterpene cyclization pathway [28]. The full biosynthetic route to these metabolites from three precursors (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), has been recently described and highlights the key role of those VHPO enzymes [29].…”

“…Comprehensive analysis of 1D and 2D NMR data of The relative stereochemistry of the dihydropyran ring of compound 1 (Figure 3) was assigned by interpretation of ROESY data ( Figure S7) and the coupling constants observed in its 1 H NMR spectrum (Table 1, Figure S2) in combination with molecular modelling using Chem3D 12.0 Pro. The biosynthetic route for all napyradiomycins described to date was also considered [3,28,29]. The almost equally intense ROESY correlations observed between H-3 and both geminal methyl groups (C-20 and C-21) together with a coupling constant value of 6.4 Hz between protons H-3 and H-4 ( Figure 3), in good agreement with a dihedral angle of 28.7° measured in the energy-minimized molecular model ( Figure S8), confirmed the relative configuration at C-3 of the dihydropyran ring in 1.…”

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

“…[97] Using a heterologous soluble expression of NapH4 in Streptomyces lividans TK23, it was shown that NapH4 is involved in the halogenation-induced cyclization of a geranyl moiety to form an exomethylene-containing chlorinated cyclohexane ring of napyradiomycin B1. [98] Though napH3 has above 50% sequence identity with NapH1 and napH4, the recombinant NapH3 enzyme exhibited no halogenation activity. Instead, it mediated the C4-to-C3 α-hydroxyketone rearrangement of the geranyl moiety with synthetic and pathway intermediate to form naphthomevalin 11.…”