Structure and Properties of an Engineered Transketolase from Maize

Abstract

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.Transketolase (TK) catalyzes the reversible transfer of two-carbon units from ketose phosphates to aldose phosphates (for review, see Schenk et al., 1998). In heterotrophic organisms, TK provides a link between glycolysis and the pentose phosphate pathway and provides precursors for nucleotide, aromatic amino acid, and vitamin biosynthesis. In addition, in plants, the enzyme plays a central role in the Calvin cycle and thereby participates in the fixation of approximately 5 ϫ 10 11 metric tons of carbon dioxide per year (Hess, 1999).TKs from a wide variety of organisms show significant sequence similarity (Schenk et al., 1997). Thus, the enzymes of man and Escherichia coli comprise about 24% identical amino acid residues. Typically, the enzymes are homodimers of 70-to 74-kD subunits. They require thiamine pyrophosphate (TPP) and divalent cations, e.g. Mg 2ϩ , for activity.TKs of animals and fungi are located in the cytoplasmic compartment. On the other hand, the TK activity in photosynthetic and in non-photosynthetic plant tissues seems to be restricted to the plastid (Schnarrenberger et al., 1995; Debnam and Emes, 1999; Henkes et al., 2001). The TKs of the plants Craterostigma plantagineum (Bernaccia et al., 1995) and pepper (Capsicum annuum; Bouvier et al., 1998) show 47% and 53% identical amino acid residues, respectively, as compared with TK from yeast. Except for two non-constitutively expressed isoforms in the resurrection plant C. plantagineum (Bernaccia et al., 1995), the plant TK genes studied to date specify N-terminal plastid-targeting sequences (Flechner et al., 1996; Bouvier et al., 1998; Henkes et al., 2001). The in vitro import of a TK precursor in isolated spinach (Spinacia oleracea) chloroplasts has been shown (Flechner et al., 1996), as well as the association of the mature TK with the thylakoid membrane of spinach chloroplasts (Teige et al., 1998).The activity of plastid TK is a limiting factor for the maximum rate of photosynthesis. A reduced level of TK activity is conducive to a reduction of the primary and secondary metabolism of tobacco (Nicotiana tabacum; Henkes et al., 2001). A reduction of TK activity also leads to decreased growth and decreased levels of aromatic acids and compounds of the phenylpropane metabolism. Due to its cru...

Subcellular localization and enzymatic properties of differentially expressed transketolase genes isolated from the desiccation tolerant resurrection plant Craterostigma plantagineum

“…There are some uncertainties about the localization of the oxidative pentose phosphate pathway, which takes place in the cytoplasm in animal cells and fungi, whereas the complete pathway in plants is only localized to chloroplasts according to Schnarrenberger et al (1995), Debnam and Emes (1999), and Henkes et al (2001). All plant transketolase enzymes of the Calvin cycle and the oxidative pentose phosphate pathway reported so far are nuclear encoded, and transported into the chloroplasts (Flechner et al 1996;Teige et al 1998;Henkes et al 2001;Gerhardt et al 2003). Besides the transketolases of the Calvin cycle and the oxidative pentose phosphate cycle, transketolases involved in the isoprenoid biosynthesis have been isolated from peppermint and pepper; these transketolases deWne a unique family conserved between bacteria and plants, but are absent in animals (Bouvier et al1998;Lange et al 1998).…”

Structural stability of E. coli transketolase to temperature and pH denaturation

“…The structure (Gerhardt et al, 2003;Littlechild et al, 1995;Nikkola et al, 1994;Veitch et al, 2004) and catalytic mechanism (Datta and Racker, 1961;de la Haba et al, 1955;Fiedler et al, 2002;Meshalkina et al, 1997;Nilsson et al, 1997;Schorken and Sprenger, 1998), of transketolases have been extensively characterised. Each monomer of TK contains a pyrophosphate-binding (PP), pyridinium-binding (Pyr) and C-terminal domain ( Fig.…”

Homology modeling of human Transketolase: Description of critical sites useful for drug design and study of the cofactor binding mode

“…A lot of work has been devoted to yeast (Saccharomyces cerevisiae), Escherichia coli and maize Transketolases and their structures have been solved by X-ray diffraction [7][8][9][10][11], revealing important aspects of the functional flexibility, metabolic profile and substrate binding of these variants. Interestingly, three distinct domains were identified in yeast Transketolase: the N-terminal, middle and C-terminal domains [8,9].…”