Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species.
An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles.
Liscombe
David K
DK
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Ziegler
Jörg
J
Schmidt
Jürgen
J
Ammer
Christian
C
Facchini
Peter J
PJ
D044182:Benzylisoquinolines / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D031675:Eschscholzia / Q000201:enzymology* / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D023281:Genomics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015394:Molecular Structure; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D031949:Thalictrum / Q000201:enzymology* / Q000235:genetics
D044182:Benzylisoquinolines; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D008780:Methyltransferases