Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

Since plants are sessile organisms, they produce a diverse range of defense chemicals, known as specialized metabolites, that contribute to the adaptation to their ecological niches (Pichersky and Lewinsohn, 2011). Chemical compounds are important for plants, as they can serve as attractants for insect pollinators or as defense against pathogens and herbivores (Pichersky and Gang, 2000). Many plant species have been used in traditional medicines for the treatment of various human diseases (Tang and Eisenbrand, 1992). Almost one-fourth of modern medicines are derived from natural sources (De Luca et al., 2012). Alkaloids are one of the most important specialized metabolites and are mostly derived from amino acids. Alkaloids display a vast variety of biological activities, and many of them are currently used for clinical purposes; examples include morphine as an analgesic, artemisinin as an antimalarial, and camptothecin as an antineoplastic (De Luca et al., 2012).

Lycopodium alkaloids (LAs) are Lys-derived alkaloids that have quinolizine or pyridine and α-pyridine nuclei in their structures (Ma and Gang, 2004). LAs have been isolated primarily from the genera Lycopodium and Huperzia, which are clubmosses (Ma and Gang, 2004). Whole plants from the families Huperziaceae and Lycopodiaceae have been used in Chinese folk medicine for the treatment of various symptoms (Ma et al., 2007). Huperzia serrata produces huperzine A (HupA), a promising candidate drug for the treatment of Alzheimer’s disease, owing to its function as a potent acetylcholinesterase inhibitor (Wang et al., 2009; Qian and Ke, 2014). HupA and its derivative ZT-1 have been evaluated in clinical trials for the treatment of Alzheimer’s disease (Ma et al., 2007; Jia et al., 2013).

Owing to the difficulties in cultivation and in vitro propagation, the biosynthetic pathways for LAs are not well documented and have been proposed based on tracer experiments using labeled precursors and plants in their natural habitats (Ma and Gang, 2004, and refs. therein). Lysine decarboxylase (LDC) has been proposed as the entry-point enzyme in the LA biosynthetic pathway, which catalyzes the decarboxylation of Lys to yield cadaverine (Fig. 1). Cadaverine is then catalyzed by CuAO to produce 5-aminopentanal, which is spontaneously cyclized to the first intermediate for LA production, Δ^{-piperideine (}Ma and Gang, 2004). Based on analyses of the EST data from LA-producing plants, several candidate genes for LA biosynthesis have been proposed; however, no further investigation has been performed (Luo et al., 2010a, 2010b). Recently, the CuAO gene from H. serrata was cloned and characterized, using degenerate primers based on the conserved sequences of the known plant CuAO enzymes; however, the cloned CuAO showed a broad substrate specificity (Sun et al., 2012).

Figure 1.

Putative biosynthetic pathway for LAs. Dotted arrows indicate more than one catalytic conversion. CuAO, Copper amine oxidase.

Recently, we showed that bifunctional lysine/ornithine decarboxylases (L/ODCs) in the Lys-derived quinolizidine alkaloid (QA)-producing legumes were recruited by the ubiquitous enzyme ornithine decarboxylase (ODC; Bunsupa et al., 2012a). ODC catalyzes the decarboxylation of l-Orn to yield putrescine, which is the main precursor for the production of Orn-derived alkaloids. In plant cells, putrescine and its derivative polyamines, spermidine and spermine, are essential for a wide range of biological processes during plant growth and development (Fuell et al., 2010). In addition to its role in alkaloid biosynthesis, cadaverine has been implicated as a growth regulator and stress-response compound in several plant species (Tomar et al., 2013).

In this study, in order to elucidate the biosynthetic pathway of LAs and the evolution of plant LDC, we cloned L/ODC from Lycopodium clavatum and H. serrata. We provide results from both in vitro and in vivo experiments to confirm the functions of L/ODC in L. clavatum. Using the tests for positive selection and assays of enzyme function, we then show the convergent evolution of plant LDC in the Lys-derived alkaloid-producing plants. Furthermore, we were able to detect the substitution site that is under positive selection and is important for improving the LDC function.

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