Primary structure of spinach‐chloroplast thioredoxin f

Abstract

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12564 Da and a molar absorption coefficient at 280 nm of 17700 M−1 cm−1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin. A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in λgt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably consitutes the N‐terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5‐kDa protein.

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

“…Based on a comparison with the published sequences of spinach (Wedel et al, 1992) and Chlarrrydomonas reiizhardtii (Stein et al, 1995) Trx m, this clone encodes for a full 60-amino acid transit peptide and for a Trx m with 112 amino acids and a size of 12,500 D. The precursor peptide is in the range of the 67 residues of the transit peptide of spinach Trx m (Wedel et al, 1992), but it is shorter than those of Trxsffrom pea (Lepiniec et al, 1992) and spinach (Kamo et al, 1989), which show 73 and 77 residues, respectively. As occurs with the transit peptides of spinach Trxs f and m, and with the transit peptide of pea Trxf, that of Trx m from pea starts with an MA motif in the N terminus; in addition, it shows a high content of (Wedel et al, 1992), pea Trx f (Lepiniec et al, 1992), spinach Trx r~ (Kamo et al, 1989), E. co//Trx (Hoog et al, 1985), and C. reinhardtii Trx m (Stein et al, 1995). The position of the a-helices and /3-strands corresponds to those of E. coliJrx (Eklund et al, 1991).…”

Modification of the reactivity of spinach chloroplast thioredoxin f by site-directed mutagenesis

“…The C73S mutation resulted in a larger loss of activity than the C73A mutation. This is somewhat surprising since the C73S mutation is rather conservative and should yield a protein significance with respect to the N-terminus of native spinach thioredoxin f. Its N-terminal residue was found to be a blocked methionine [11]. The N-terminal methionine could be Met1 or Met10 in Fig.…”

“…Thioredoxin f has been purified from spinach [7 -9] and pea [10] chloroplasts and its primary structure determined [11]. It has been cloned [11,12] and overexpressed in Escherichia coli [13] and its crystal structure has recently been solved [14].…”

“…It has been cloned [11,12] and overexpressed in Escherichia coli [13] and its crystal structure has recently been solved [14]. Thioredoxin f, with a molecular mass of 13 500, has beside the active site sequence TrpCys-Gly-Pro-Cys, common to most thioredoxins, relatively little resemblance with the second chloroplast thioredoxin sharing only 30% residue identity.…”

A novel thioredoxin‐like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants

“…Plant and E. coli thioredoxin sequence names correspond to SWISS-PROT identification names. THII_TOBAC: tobacco thioredoxin h 2 (Brugidou et al, 1993); THIH_TOBAC: tobacco thioredoxin h 1 (Brugidou et al, 1993); THIF_SPIOL: spinach thioredoxin f (Kamo et al, 1989); THIM_SPIOL: spinach thioredoxin m (Maeda et al, 1986); THIO_ECOLI: E. coli thioredoxin (Hö ö g et al, 1985). (c) Sequence alignment of CDSP 32 N-terminal 121 residues and C-terminal 122 residues.…”