Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis.
Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (+)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9-10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (+)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (+)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (+)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (+)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.
Larbat
Romain
R
UMR 1121 Agronomie Environment INPL-INRA, ENSAIA, Vandoeuvre-lès-Nancy, France.
Kellner
Sandra
S
Specker
Silvia
S
Hehn
Alain
A
Gontier
Eric
E
Hans
Joachim
J
Bourgaud
Frederic
F
Matern
Ulrich
U
D000595:Amino Acid Sequence; D031165:Ammi / Q000201:enzymology*; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D011564:Furocoumarins / Q000096:biosynthesis*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014468:Umbelliferones / Q000737:chemistry
D011564:Furocoumarins; D010940:Plant Proteins; D014468:Umbelliferones; C031477:7-hydroxycoumarin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C050853:psoralen synthase