Structure and Properties of an Engineered Transketolase from Maize1

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.

[1]  F. Sanger,et al.  A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. , 1975, Journal of molecular biology.

[2]  D I Svergun,et al.  Crystal Versus Solution Structures of Thiamine Diphosphate-dependent Enzymes* , 2000, The Journal of Biological Chemistry.

[3]  M Wilm,et al.  Electrospray mass spectrometry for protein characterization. , 1995, Trends in biochemical sciences.

[4]  R. Duggleby,et al.  Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase , 1997, Journal of Molecular Evolution.

[5]  F. Salamini,et al.  The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during the rehydration phase. , 1995, The EMBO journal.

[6]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[7]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[8]  J. Pletcher,et al.  Crystal and molecular structure of thiamine pyrophosphate hydrochloride. , 1972, Journal of the American Chemical Society.

[9]  M. Teige,et al.  Purification, properties and in situ localization of the amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase from spinach chloroplasts. , 1998, European journal of biochemistry.

[10]  G. Schneider,et al.  Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis. , 1997, European journal of biochemistry.

[11]  G. Schneider,et al.  Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate. , 1994, The Journal of biological chemistry.

[12]  G. N. Ramachandran,et al.  Conformation of polypeptides and proteins. , 1968, Advances in protein chemistry.

[13]  W. Martin,et al.  Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves , 1995, Plant physiology.

[14]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[15]  G. Schneider,et al.  Three‐dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution. , 1992, The EMBO journal.

[16]  R. Huber,et al.  Accurate Bond and Angle Parameters for X-ray Protein Structure Refinement , 1991 .

[17]  P. Heinrich,et al.  Studies on the reconstitution of apotransketolase with thiamine pyrophosphate and analogs of the coenzyme. , 1972, European journal of biochemistry.

[18]  G. Schneider,et al.  Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Martin,et al.  Aspartate 155 of human transketolase is essential for thiamine diphosphate-magnesium binding, and cofactor binding is required for dimer formation. , 1997, Biochimica et biophysica acta.

[20]  M. Stitt,et al.  A Small Decrease of Plastid Transketolase Activity in Antisense Tobacco Transformants Has Dramatic Effects on Photosynthesis and Phenylpropanoid Metabolism , 2001, Plant Cell.

[21]  G. Schneider,et al.  Examination of Substrate Binding in Thiamin Diphosphate- dependent Transketolase by Protein Crystallography and Site-directed Mutagenesis* , 1997, The Journal of Biological Chemistry.

[22]  R. Backhaus,et al.  Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits1. , 1998, Plant physiology.

[23]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.

[24]  J. Villafranca,et al.  Heptulose synthesis from nonphosphorylated aldoses and ketoses by spinach transketolase. , 1971, The Journal of biological chemistry.

[25]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[26]  G. Schneider,et al.  Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 Å resolution , 1993, FEBS letters.

[27]  W. Martin,et al.  Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts , 1996, Plant Molecular Biology.

[28]  D. Blow,et al.  The detection of sub‐units within the crystallographic asymmetric unit , 1962 .

[29]  G. Schneider,et al.  Crystallography and mutagenesis of transketolase: mechanistic implications for enzymatic thiamin catalysis. , 1998, Biochimica et biophysica acta.

[30]  Robert Huber,et al.  Die automatisierte Faltmolekülmethode , 1965 .

[31]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[32]  A. Paterson,et al.  The molecular basis of C4 photosynthesis in sorghum: isolation, characterization and RFLP mapping of mesophyll- and bundle-sheath-specific cDNAs obtained by differential screening , 1998, Plant Molecular Biology.

[33]  G. Schneider,et al.  Refined structure of transketolase from Saccharomyces cerevisiae at 2.0 A resolution. , 1994, Journal of molecular biology.

[34]  R. Duggleby,et al.  Properties and functions of the thiamin diphosphate dependent enzyme transketolase. , 1998, The international journal of biochemistry & cell biology.

[35]  Michael J. Emes,et al.  Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues , 1999 .

[36]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[37]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[38]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[39]  G. Schneider,et al.  A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. , 1993, Structure.