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Plant Physiol. 2016 Aug;171(4):2432-44. doi: 10.1104/pp.16.00639. Epub 2016 Jun 14.

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

Author information

1
Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan (S.B., A.M., K.A., K.K., H.U., Mad.Y., K.S., Mam.Y.);Faculty of Pharmacy, Mahidol University, Ratchathewi, Bangkok 10400, Thailand (S.B.);Kyushu Institute of Technology, Iizuka-shi, Fukuoka 820-8502, Japan (K.H.);RIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama 230-0045, Japan (R.S., A.O., K.S.); andFaculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan (A.O.).
2
Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan (S.B., A.M., K.A., K.K., H.U., Mad.Y., K.S., Mam.Y.);Faculty of Pharmacy, Mahidol University, Ratchathewi, Bangkok 10400, Thailand (S.B.);Kyushu Institute of Technology, Iizuka-shi, Fukuoka 820-8502, Japan (K.H.);RIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama 230-0045, Japan (R.S., A.O., K.S.); andFaculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan (A.O.) ksaito@faculty.chiba-u.jp mamiy@faculty.chiba-u.jp.

Abstract

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

PMID:
27303024
PMCID:
PMC4972286
DOI:
10.1104/pp.16.00639
[Indexed for MEDLINE]
Free PMC Article

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