Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii

Abstract

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe 2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs

“…The kinetics for 1 under determined optimal conditions gave a K m = 70.2 ± 4.0 μM and k cat = 0.58 ± 0.01 s −1 (Figures S30 and S31), which suggests comparable overall catalytic efficiencies of DolJ and LynB7. 23 The diastereomeric analogue 2 was not active in the DolJ reaction (Figure 3A,B). We also prepared recombinant BarH (Figure S7), and it showed the same specificity toward compounds 1 and 2 as DolJ (Figure 3B).…”

“…The product might then be oxidatively decarboxylated, potentially by DolJ, to result in Dol-10's final thiazole (Figure 2B). LynB7, which shares 78% identity to DolJ, was recently shown to mediate the same transformation in lyngbyapeptin B biosynthesis, 23 which may also occur in barbamide biosynthesis by BarH. 22 DolJ, BarH, and LynB7 are members of the nonheme diiron monooxygenase family (Figure S6), 23 which catalyzes a range of oxidation reactions (e.g., hydroxylation).…”

“…LynB7, which shares 78% identity to DolJ, was recently shown to mediate the same transformation in lyngbyapeptin B biosynthesis, 23 which may also occur in barbamide biosynthesis by BarH. 22 DolJ, BarH, and LynB7 are members of the nonheme diiron monooxygenase family (Figure S6), 23 which catalyzes a range of oxidation reactions (e.g., hydroxylation). 24 A subsequent gene, in the opposite direction to dolJ, encodes a cyclase (Orf11) succeeded by a presumed arsenite transporter (Orf12).…”

Bioinformatic Discovery of a Cambialistic Monooxygenase

“…Recently, we demonstrated that AibH1H2 is a Mn/Fe-dependent monooxygenase that hydroxylates an aliphatic C–H bond of 2-aminoisobutyric acid . The protein components of AibH1H2 stem from a largely uncharacterized structural superfamily, PF04909, containing over 100,000 unique proteins that can individually function as either carboxylases, hydratases, hydrolases, or monooxygenases. − Representatives of the latter enzyme classcollectively defined here as “amidohydrolase-related dinuclear oxygenases” (AROs)include PtmU3 and AibH2, , which employ dinuclear cofactors with different metal identities. In contrast, the other characterized PF04909 enzymes either lack a cofactor or employ a mononuclear metal site that can be Mn or Zn. , The primary structural features that differentiate mono- versus dinuclear active sites, engender monooxygenase functionality, and control the metalation of AibH2 and PtmU3 are unknown.…”

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“…Genome mining of the cyanobacterium Moorena bouillonii has revealed the enzymes involved in the biosynthesis of the hybrid PKS-nonribosomal peptide lyngbyapeptin B 24 . 22 This work allowed characterisation of the O -methyltransferase domain from the LynB2 PKS module, which generates the ( E )-3-methoxy-2-butenoyl starter unit that feeds nonribosomal peptide assembly. Various 2,3-diaminopropionates have been synthesised and investigated as alternative precursors during monobactam biosynthesis.…”