Molecular characterization of berberine bridge enzyme genes from opium poppy.
In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.
Facchini
P J
PJ
Department of Biological Sciences, University of Calgary, Alberta, Canada. pfacchin@acs.ucalgary.ca
Penzes
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Johnson
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Bull
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D000595:Amino Acid Sequence; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics*; D010944:Plants / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
C071690:reticuline oxidase; D010089:Oxidoreductases, N-Demethylating