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.
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.
McMillan
L K
LK
Centre for Functional Genomics, Institute of Molecular BioSciences, College of Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
Carr
R L
RL
Young
C A
CA
Astin
J W
JW
Lowe
R G T
RG
Parker
E J
EJ
Jameson
G B
GB
Finch
S C
SC
Miles
C O
CO
McManus
O B
OB
Schmalhofer
W A
WA
Garcia
M L
ML
Kaczorowski
G J
GJ
Goetz
M
M
Tkacz
J S
JS
Scott
B
B
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007211:Indoles / Q000378:metabolism* / Q000494:pharmacology*; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008322:Mammals; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007211:Indoles; D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011994:Recombinant Proteins; C048221:paspaline; C048220:paxilline; D003577:Cytochrome P-450 Enzyme System