Metabolic Characterization of Hyoscyamus niger Ornithine Decarboxylase.

Tengfei Zhao,Xiaozhong Lan,Siqi Li,Feng Bai,Chunxian Yang,Ge Bai,Min Chen,Zhihua Liao,Fangyuan Zhang,Changjian Wang

doi:10.3389/fpls.2019.00229

Tengfei Zhao, Xiaozhong Lan + Show 8 more

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https://doi.org/10.3389/fpls.2019.00229

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Abstract

Ornithine decarboxylase (ODC) catalyzes ornithine decarboxylation to yield putrescine, a key precursor of polyamines, and tropane alkaloids (TAs). Here, to investigate in depth the role of ODC in polyamine/TA biosynthesis and to provide a candidate gene for engineering polyamine/TA production, the ODC gene (HnODC) was characterized from Hyoscyamus niger, a TA-producing plant. Our phylogenetic analysis revealed that HnODC was clustered with ODC enzymes of plants. Experimental work showed HnODC highly expressed in H. niger roots and induced by methyl jasmonate (MeJA). In the MeJA treatment, the production of both putrescine and N-methylputrescine were markedly promoted in roots, while contents of putrescine, spermidine, and spermine were all significantly increased in leaves. By contrast, MeJA did not significantly change the production of either hyoscyamine or scopolamine in H. niger plants. Building on these results, the 50-kDa His-tagged HnODC proteins were purified for enzymatic assays. When ornithine was fed to HnODC, the putrescine product was detected by HPLC, indicating HnODC catalyzed ornithine to form putrescine. Finally, we also investigated the enzymatic kinetics of HnODC. Its Km, Vmax, and Kcat values for ornithine were respectively 2.62 ± 0.11 mM, 1.87 ± 0.023 nmol min-1 μg-1 and 1.57 ± 0.015 s-1, at pH 8.0 and at 30°C. The HnODC enzyme displays a much higher catalytic efficiency than most reported plant ODCs, suggesting it may be an ideal candidate gene for engineering polyamine/TA biosynthesis.

Highlights

Polyamines, including putrescine, spermidine, and spermine, are involved in many important biological processes of plants, such as their growth, development, and adaption to biotic and abiotic stresses (Kusano et al, 2008; Kusano and Suzuki, 2015; Aloisi et al, 2016)
The BLASTP analysis indicated HnODC belonged to the superfamily of type III pyridoxal phosphate (PLP)-dependent enzymes, and that it resembled the ornithine decarboxylase (ODC) in GenBank
All ODCs contain the conserved PLP-binding motifs composed of PFYAVKCN, and the GPTCD sequences, both of which are necessary for ODC activity (Coleman et al, 2003)

Summary

Introduction

Polyamines, including putrescine, spermidine, and spermine, are involved in many important biological processes of plants, such as their growth, development, and adaption to biotic and abiotic stresses (Kusano et al, 2008; Kusano and Suzuki, 2015; Aloisi et al, 2016). Putrescine is essential for the synthesis of polyamines and putrescine-derived alkaloids (Jirschitzka et al, 2012), because forming putrescine is the first step in the polyamines biosynthetic pathway (Figure 1), providing a ley precursor for spermine and spermidine (Kusano and Suzuki, 2015). Putrescine can become methylated to form N-methylputrescine, a key intermediate compound of nicotine and Metabolic Roles of HnODC pharmaceutical tropane alkaloids (TAs) (Biastoff et al, 2009). We may speculate that ODC rather than ADC participates in the biosynthesis of putrescinederived alkaloids. To resolve this issue clearly requires further experimental investigation

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