A PLP-Dependent Polyketide Chain Releasing Mechanism in the Biosynthesis of Mycotoxin Fumonisins inFusarium verticillioides

Abstract

Fumonisins are polyketide-derived mycotoxins produced by several plant pathogenic fungi. The toxins cause several fatal diseases in domestic animals and are associated with esophageal cancer and neural tube defects in humans. Fumonisins contain a highly reduced, acyclic 18-carbon chain, which is synthesized by an iterative polyketide synthase (PKS). This PKS does not contain a thioesterase or cyclase domain that is found in other PKSs for the release of the covalently linked polyketide chain. In this study, we expressed the acyl carrier protein (ACP) of FUM1 and in vitro loaded acyl chains to the ACP from acyl-CoA using a promiscuous 4'-phosphopantetheinyl transferase. We then expressed FUM8, which is homologous to 2-oxoamine synthase genes, in yeast and showed that the enzyme is able to offload the acyl chains from ACP. Products resulted from the decarboxylative condensation between l-alanine and acyl-S-ACP were detected by GC-MS. The enzyme activity was dependent on pyridoxal 5'-phosphate (PLP), and C18-S-ACP was the preferred substrate. The results revealed a novel polyketide chain-releasing mechanism, in which a PLP-dependent enzyme catalyzes the termination and offloading of the polyketide chain as well as the introduction of a new carbon-carbon bond and an amino group to the chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain release found in bacterial and other fungal polyketide biosyntheses.

Identification of Early Fumonisin Biosynthetic Intermediates by Inactivation of the FUM6 Gene in Fusarium verticillioides

“…The chemical shift of the quaternary carbon falls into the range expected for a hydroxylated carbon. Using a mixing time (d9) of 80 or 160 ms for acquisition of HSQC-TOCSY also revealed a correlation from the terminal H-20 at 0.86 ppm to a 13 well as the C-/H-22 methyl group showed chemical shifts nearly identical to those of 7. However, the chemical shifts of C/H-1−C/H-3 were affected by the hydroxylation 13−15 bonds away and had generally shifted to slightly lower 1 H chemical shifts (maximum −0.16 ppm for H-2) and slightly higher 13 C chemical shifts (maximum +2.2 ppm for C-1).…”

“…In addition, 10,14-dimethyloctadecanoic acid may exist only as an enzyme-bound intermediate. 13 In the proposed pathway, 2-amino-12,16-dimethylicosane-3-one undergoes hydroxylation at C-14 and C-15 followed by esterification of six-carbon tricarboxylate moieties to the oxygen atoms at C-14 and C-15. 14 The resulting fumonisin analogue then undergoes reduction of the C-3 carbonyl to form a C-3 hydroxyl, as well as hydroxylation at C-5 and/or C-10 ( Figure 2).…”

Structural insights into nonribosomal peptide enzymatic assembly lines

“…The termination modules for PKS, FAS and NRPS typically contain a thioesterase (TE) domain as the most downstream domain and catalyze the disconnection of the full length acyl/peptidyl chain from the adjacent downstream ACP/PCP domain 2, 16, 17, 40, 47-51. TE domains can act as hydrolases, reductases, or regiospecific macrocyclization catalysts and can release the PK, FA or NRP nascent product 52-66. A variety of tailoring enzymes can work on the acyl or peptidyl chain.…”

Surveys of non-ribosomal peptide and polyketide assembly lines in fungi and prospects for their analysis in vitro and in vivo

“…Termination was identified to proceed via PLP dependent decarboxylative condensation of L -alanine and acyl- S -ACP with greatest product formation observed for C 18 - S -ACP. This study by Gerber et al discloses a new highly reducing IPKS termination mechanism catalyzed by non-standard thioesterase/cyclase chain release enzymes [60]. Utilizing contemporary mass spectrometry, this study contributes to the specific knowledge of fumonisin biosynthesis and the general mechanisms possible for thiotemplate transformations.…”