Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels

Abstract

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

Identification of Two Aflatrem Biosynthesis Gene Loci in Aspergillus flavus and Metabolic Engineering of Penicillium paxilli To Elucidate Their Function

“…atmP and atmQ are both predicted to encode cytochrome P450 monooxygenases and are putative orthologs of paxP and paxQ, respectively. The products of each of these genes contained all of the functional domains expected for (11,22). atmP contained five introns identical in the same relative positions as the introns in paxP, and the gene products shared 61% identity (70% similarity).…”

“…By analogy with the pathway for paxilline biosynthesis in P. paxilli (22,28,29,41), the identification of biosynthesis intermediates by LC-MS, and genetic complementation experiments involving atmM, atmP, and atmQ, a proposed pathway for aflatrem biosynthesis in A. flavus is presented in Fig. 8.…”

“…Cultures of A. flavus strains NRRL6541 and NRRL3357 were maintained on 2.4% Difco potato dextrose agar (Becton Dickinson, Maryland) plates or as spore suspensions in 10% (vol/vol) glycerol at Ϫ80°C. For aflatrem production, 100-ml Erlenmeyer flasks containing 25 ml of YEPGA medium (22) were inoculated with 5 ϫ 10 6 spores and grown with shaking (200 rpm) for 48 h at 30°C. Aliquots of 2 ml were used to inoculate 250-ml Erlenmeyer flasks containing 50 ml of aflatrem production medium (44), which were grown without agitation at 29°C in the dark until harvesting.…”

“…The majority of metabolites in this group share a common structural core consisting of a tetracyclic diterpene derived from geranylgeranyldiphosphate (GGPP) (7), fused to an indole moiety, most likely derived from indole 3-glycerol phosphate (5). These precursors combine to form paspaline, the first stable indole-diterpene required for paxilline biosynthesis (22,28,29). More complex indole-diterpenes generally consist of a paxilline-like core modified with a variety of decorations including different patterns of prenylation, hydroxylation, epoxidation, acetylation, and ring rearrangements (24).…”

“…The products of four of these genes, a GGPP synthase (PaxG), a prenyltransferase (PaxC), a flavin adenine dinucleotide (FAD)-dependent monooxygenase (PaxM), and a protein of unknown function (PaxB), are all required for the biosynthesis of paspaline (29). This product is subsequently converted to paxilline by the sequential action of two cytochrome P450 monooxygenases, PaxP and PaxQ (22,28). The structural similarity of paxilline and aflatrem ( Fig.…”