FtmOx1, a non-heme Fe(ii) and α-ketoglutarate-dependent dioxygenase, catalyses the endoperoxide formation of verruculogen in Aspergillus fumigatus

Abstract

Verruculogen is a tremorgenic mycotoxin and contains an endoperoxide bond. In this study, we describe the cloning, overexpression and purification of a non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase FtmOx1 from Aspergillus fumigatus, which catalyses the conversion of fumitremorgin B to verruculogen by inserting an endoperoxide bond between two prenyl moieties. Incubation with (18)O(2)-enriched atmosphere demonstrated that both oxygen atoms of the endoperoxide bond are derived from one molecule of O(2). FtmOx1 is the first endoperoxide-forming non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase reported so far. A mechanism of FtmOx1-catalysed verruculogen formation is postulated and discussed.

Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes

“…Using recombinant enzymes, we have demonstrated that FtmPT2/FtmH catalyzes the prenylation of 12,13-dihydroxyfumitremorgin C in the presence of dimethylallyl diphosphate, resulting in the formation of fumitremorgin B, 35 which is then converted to verruculogen by FtmOx1/FtmF by inserting an endoperoxide bond between the two prenyl moieties. 32 We have shown that FtmOx1 reaction was absolutely dependent on the presence of Fe(II) and a-ketoglutarate, and therefore functions as a non-heme Fe(II) and a-ketoglutaratedependent dioxygenase. Known a-ketoglutarate-dependent dioxygenases usually transfer one oxygen atom to the substrate and another to a-ketoglutarate resulting in the formation of succinate.…”

“…36 In contrast, both oxygen atoms from a single O 2 molecule were incorporated into the structure of verruculogen, which was proven by incubation of fumitremorgin B with FtmOx1 in 18 O 2 enriched atmosphere. 32 Therefore, FtmOx1 represents the first endoperoxide forming non-heme Fe(II) and a-ketoglutarate-dependent dioxygenase reported so far. Until now, functions of two genes ftmMT/ftmD and ftmO/ftmI have to be proven experimentally.…”

“…Furthermore, they have same order and orientation in these clusters. The end product of this cluster was proven to be verruculogen (11) (see below), 32 but it could not be excluded that fumitremorgin A (10) is the true end product instead. The fumitremorgin/verruculogen cluster was very likely not expressed in A. fumigatus Af293; therefore, no secondary metabolites of this cluster could be detected in this strain.…”

“…27,31,32,35 In total, functions of seven genes have been proven experimentally. On the basis of the obtained bioinformatic, molecular biological and biochemical results, the following biosynthetic pathway has been postulated ( Figure 3).…”

Chemical Biology: What is Its Role in Drug Discovery?

“…The stereoselective synthesis of deuterated analogues of Lglutamate was accomplished by Schofield and the analogues used in a study on carbapenem biosynthesis ( Figure 6B) (Ducho et al, 2009). A report on the first -ketoglutarate-dependant dioxygenase with non-heme Fe(II) to catalyse the formation of an endoperoxide bond has appeared (Steffan et al, 2009). This enzyme was shown to convert fumitremorgin B to verruculogen by the insertion of an endoperoxide bond using both oxygen atoms of O 2 .…”