On the Sequence of Bond Formation in Loline Alkaloid Biosynthesis

Abstract

Loline alkaloids are saturated pyrrolizidines with an oxygen bridge between carbon atoms C-2 and C-7 and an amino group on C-1. They are bioprotective alkaloids produced by Epichloë and Neotyphodium species, mutualistic fungal endophytes that are symbiotic with cool-season grasses. The sequence of bond formation in loline alkaloid biosynthesis was determined by synthesizing deuterated forms of potential intermediates and feeding them to cultures of the endophyte Neotyphodium uncinatum. These cultures incorporated deuterium from labeled N-(3-amino-3-carboxypropyl)proline and exo-1-aminopyrrolizidine into N-formylloline. The first result suggests that N-(3-amino-3-carboxypropyl)proline is the first committed intermediate in loline biosynthesis, and the second result demonstrates that the pyrrolizidine rings form before the ether bridge. The incorporation of these two compounds into lolines and the lack of incorporation of several related compounds clarify the order of bond formation in loline alkaloid biosynthesis.

Regulation of a Chemical Defense against Herbivory Produced by Symbiotic Fungi in Grass Plants

“…Translocation of Asn and Gln transports fixed nitrogen from older to younger tissues (Nakano et al, 2000). The lolines are derived from the Gln derivative, Pro, and the Asnderivative, L-homoserine (Hse), probably via conjugation of its derivative, O-acetyl-Hse, to the ring nitrogen atom of Pro (Blankenship et al, 2005;Faulkner et al, 2006). In fungi, O-acetyl-Hse is the precursor of Thr, Ile, and Met, whereas in plants, O-phospho-Hse plays the same role.…”

“…Furthermore, elevated LOL gene expression in MF-N. uncinatum center leaves corresponded with higher expression of the two amino acid biosynthesis genes analyzed: proC for synthesis of Pro and metE for synthesis of O-acetylhomoserine, the intermediate in pathways to Met, Ile, Thr, and lolines. Nevertheless, isotopically labeled precursor amino acids, Glu, Pro, Orn, Asp, and Hse, are readily taken up by N. uncinatum in minimal medium culture and incorporated into lolines (Blankenship et al, 2005;Faulkner et al, 2006). Thus, the LA biosynthetic pathway makes use of free amino acids that are specifically mobilized to younger plant tissues.…”

“…Neotyphodium uncinatum contains two highly similar LOL clusters (LOL1 and LOL2), and a single LOL cluster has been found in each of the LA-producing species, Neotyphodium coenophialum, Neotyphodium siegelii, and some strains of Epichloë festucae, among others (Spiering et al, 2005;Kutil et al, 2007). Fermentation cultures of N. uncinatum produce lolines, and studies involving application of labeled precursors and intermediates have almost completely elucidated the LA biosynthetic pathway (Blankenship et al, 2005;Spiering et al, 2005;Faulkner et al, 2006;Schardl et al, 2007). Putative roles of the LOL gene products-based on sequence relationships to known enzyme classes-fit well with the pathway.…”

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

“…Results from applying isotopically labeled amino acids or putative intermediates to these loline-producing cultures have identified precursor amino acids (Blankenship et al, 2005) and biosynthetic intermediates (Faulkner et al, 2006), and indicate that loline biosynthesis follows a novel pathway different from the necine biosynthesis pathway (Hartmann and Witte, 1995). The loline precursor, L-homoserine (Hse) (probably in the form of O-acetylHse) is condensed with the secondary amine in L-proline (Pro) (Blankenship et al, 2005), giving N-(3-amino-3-carboxypropyl)proline as the first committed intermediate in the loline pathway (Faulkner et al, 2006). This c-substitution reaction is probably catalyzed by the product of the lolC gene, which is predicted to be a c-class pyridoxal phosphate (PLP)-containing enzyme (Spiering et al, 2002(Spiering et al, , 2005.…”

“…This c-substitution reaction is probably catalyzed by the product of the lolC gene, which is predicted to be a c-class pyridoxal phosphate (PLP)-containing enzyme (Spiering et al, 2002(Spiering et al, , 2005. Subsequent steps form the core pyrrolizidine ring, after which are added the ether linkage between C-2 and C-7, and methyl, acetyl, or formyl groups on the C-1 amine (Faulkner et al, 2006).…”