Umecyanin (UMC) is a type 1 copper-containing protein which originates from horseradish roots and belongs to the stellacyanin subclass of the phytocyanins, a ubiquitous family of plant cupredoxins. The crystal structures of Cu(II) and Cu(I) UMC have been determined at 1.9 and 1.8 A, respectively. The protein has an overall fold similar to those of other phytocyanins. At the active site the cupric ion is coordinated by the N(delta1) atoms of His44 and His90, the S(gamma) of Cys85, and the O(epsilon)(1) of Gln95 in a distorted tetrahedral geometry. Both His ligands are solvent exposed and are surrounded by nonpolar and polar side chains on the protein surface. Thus, UMC does not possess a distinct hydrophobic patch close to the active site in contrast to almost all other cupredoxins. UMC has a large surface acidic patch situated approximately 10-30 A from the active site. The structure of Cu(I) UMC is the first determined for a reduced phytocyanin and demonstrates that the coordination environment of the cuprous ion is more trigonal pyramidal. This subtle change in geometry is primarily due to the Cu-N(delta1)(His44) and Cu-O(epsilon1)(Gln95) bond lengths increasing from 2.0 and 2.3 A in Cu(II) UMC to 2.2 and 2.5 A, respectively, in the reduced form, as a consequence of slight rotations of the His44 and Gln95 side chains. The limited structural changes upon redox interconversion at the active site of this stellacyanin are analogous to those observed in a typical type 1 copper site with an axial Met ligand and along with its surface features suggest a role for UMC in interprotein electron transfer.

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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...