Amino Acid Sequences of Ferredoxins from Scopolia japonica and Lycium chinense: Their Similarities to That of Datura arborea

Yoshiki Mino
2002 Biological and Pharmaceutical Bulletin  
There are many examples of the use of protein characteristics in chemotaxonomy since Zuckerkandl and Pauling's seminal paper in 1965. 2) The present study is one of a series designed to provide sequence information on solanaceous ferredoxins (Fds) with the aim of elucidating the effectiveness of 'protein chemotaxonomy,' molecular taxonomy based on the primary structures of common plant proteins. We have selected Fd, one of the iron-sulfur proteins which act as electron carriers in
more » ... electron transport, for the study because Fds are widely distributed in plants, and because their small molecular size facilitates studies on their structure. I earlier reported the primary structures of Fds from seven Datura plants, [3] [4] [5] [6] Physalis alkekengi var. francheti, 7) Nicotiana tabacum, 8) and Capsicum annuum. 1) In this report, I have isolated Fds from Scopolia japonica and Lycium chinense, important medicinal drugs in China and Japan, and determined their amino acid sequences. They were also compared with primary structures of Fds of higher plants. MATERIALS AND METHODS Materials Scopolia japonica and Lycium chinense were cultivated in the herb garden at the author's university. Isolation of Ferredoxins Each protein (1.5 or 6.5 mg) was purified from the fresh leaves (0.6 or 1.0 kg) of S. japonica or L. chinense as described previously. 3, 7) Sequence Determination The amino acid sequences of the Fds were determined using a gas-phase protein sequencer by automated Edman degradation of Cm-Fds, and the peptides obtained by lysyl endopeptidase, trypsin, or endoproteinase Asp-N digestion. The C-terminal analysis was done with carboxypeptidase Y. The detailed procedure and the other methods were described in previous reports. 3, 7) Construction of Phylogenetic Tree The phylogenetic tree was constructed from the amino acid sequences (97 residues) of the higher plant Fds (24 species) using the unweighed pair-group method with arithmetical averages (UPGMA) method of Nei (GENETYX software, Software Development, Japan). 9) RESULTS AND DISCUSSION The absorption maxima in the UV-Vis spectrum of L. chinense (Lc)-Fd were at 275, 285 (sh), 330, 420, 465 nm with A max /A 275 nm ratios of 0.87, 0.65, and 0.59, respectively, for the maxima. The S. japonica (Sj)-Fd also exhibited virtually the same spectrum as Lc-Fd. These spectra were characteristic of [2Fe-2S] Fds from other higher plants. 10, 11) The sequencing strategy is summarized in Fig. 1 . The analytical results for the amino acid compositions of both Cm-Fds and the peptides obtained by enzymic digestion were consistent with the final derived sequences. Automated Edman degradation of the Lc-Cm-Fd yielded the amino-terminal sequence up to the 53rd cycle, except for several slightly doubtful amino acid residues. The lysyl endopeptidase digestion gave three short peptides (L-1 (1-4), L-2 (5-6), and L-5 (92-97)) and two long peptides (L-3 (7-50) and L-4 (51-91)). These peptides were isolated by reversed-phase HPLC; their t R s were 14.5 for L-1, 20.0 for L-5, 44.8 for L-3, and 46.4 min for L-4, while L-2 was missing. The Edman degradation of L-3-T-2, obtained by tryptic digestion of L-3 (7-50), confirmed the sequence of 41-50. L-4 and L-5 covered the sequences of 50-91 and 92-97 (C-terminal), respectively. Sequence analysis of L-4-D-6 (84-91), obtained by endoproteinase Asp-N digestion of L-4, confirmed the end part of L-4 (50-91). In solanaceous Fds tested so far, Lys-91 was insensitive to trypsin or lysyl endopeptidase cleavage probably due to the adjacent sequence, -Lys-Glu-Glu-Glu-. 8) Only for C. annuum Fd was Lys-91 sensitive to both enzymes because of the adjacent sequence, -Lys-Glu-Ala-Glu-. 1) In the case of Lc-Fd, Lys-91 in the sequence, -Lys-Glu-Glu-Ala-, was sensitive to lysyl endopeptidase, but insensitive to trypsin cleavage. These results suggest that the resistances of -Lys-Glu-Glu-Glu-, -Lys-Glu-Glu-Ala-, and -Lys-Glu-Ala-Glu-against both enzymes decrease in that order. The N-terminal sequence was confirmed by the isolation of L-1 (Ala-Thr-Tyr-Lys). Carboxypeptidase Y digestion of the Cm-Fd for different periods of time suggested the C-terminal sequence to be -Leu-Thr-Gly-COOH. This result was in good agreement with the C-terminal sequence by Edman degradation of the peptide, L-5 (92-97). The sequence analysis for Sj-Fd was con-The complete amino acid sequences of [2Fe-2S] ferredoxins from Scopolia japonica and Lycium chinense have been determined by automated Edman degradation of the entire Cm-proteins and of the peptides obtained by enzymatic digestions. These two ferredoxins exhibited only 2-7 differences in the amino acid sequence when compared to the Datura-ferredoxins (D. stramonium, D. metel, and D. arborea), and especially only 2 or 3 differences compared to D. arborea. On the contrary, 8-19 differences were observed among the other solanaceous ferredoxins. This suggests that S. japonica and L. chinense are closely related taxonomically to Datura plants, especially to D. arborea.
doi:10.1248/bpb.25.1367 pmid:12392097 fatcat:elc45hfornctxj5eglozaifp6i