In vitro characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the e...

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Published in:Organic & biomolecular chemistry 2021-03, Vol.19 (10), p.2302-2311
Main Authors: Kresna, I Dewa Made, Linares-Otoya, Luis, Milzarek, Tobias, Duell, Elke R, Mir Mohseni, Mahsa, Mettal, Ute, König, Gabriele M, Gulder, Tobias A M, Schäberle, Till F
Format: Article
Language:English
Subjects:
2,4-Dichlorophenol
Acids
Acyl carrier protein
Adenylation
Aromatic compounds
Biosynthesis
Condensates
Cytochrome
Cytochrome P450
Cytochromes P450
Enzymes
p-Hydroxybenzoic acid
Proteins
Substrates
Thioesterase
Triphenyls
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Summary:The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.
ISSN:1477-0520
1477-0539
DOI:10.1039/d0ob02372h