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McMillan LK, Carr RL, Young CA, Astin JW, Lowe RG, Parker EJ,
Jameson GB, Finch SC, Miles CO, McManus OB, Schmalhofer WA,
Garcia ML, Kaczorowski GJ, Goetz M, Tkacz JS, Scott B Mol
Genet Genomics. 2003 Oct;270(1):9-23.
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..
Centre for Functional Genomics, Institute of Molecular
BioSciences, College of Sciences, Massey University, Private Bag
11 222, Palmerston North, New Zealand.
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.
PMID: 12884010 |
