Identification of the Cysteine Residues Involved in Redox Modification of Plant Plastidic Glucose-6-phosphate Dehydrogenase

The cDNA sequences encoding cytosolic and light-modulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli. Characterization of the recombinant enzymes showed that they closely resembled their native counterparts. Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction. As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant. To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by site-directed mutagenesis. Two mutant proteins exhibited characteristics of the reduced wild-type enzyme. Exchange of either Cys149 or Cys157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH. The cDNA sequences encoding cytosolic and light-modulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli. Characterization of the recombinant enzymes showed that they closely resembled their native counterparts. Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction. As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant. To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by site-directed mutagenesis. Two mutant proteins exhibited characteristics of the reduced wild-type enzyme. Exchange of either Cys149 or Cys157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH. G6PDH 1The abbreviations used are: G6PDH, glucose-6-phosphate dehydrogenase; DTTred, reduced dithiothreitol; PCR, polymerase chain reaction; bp, base pair(s). (EC 1.1.1.49) catalyzes the first step of the oxidative pentose-phosphate pathway. The main function of the enzyme is to provide NADPH for reductive biosyntheses. In plant tissues, at least two G6PDH isoforms exist in two different compartments, one in the cytosol and one in the chloroplast stroma (1Heber U. Hudson M.A. Hallier U.W. Z. Naturforsch. 1967; 22b: 1200-1215Crossref Scopus (45) Google Scholar, 2Schnarrenberger C. Oeser A. Tolbert N.E. Arch. Biochem. Biophys. 1973; 154: 438-448Crossref PubMed Scopus (132) Google Scholar). The activity of several chloroplast enzymes is known to be regulated by reversible thiol-disulfide interchange (3Buchanan B.B. Annu. Rev. Plant Physiol. 1980; 31: 341-374Crossref Google Scholar). During photosynthetic electron transport in the light, covalent redox modification mediated by a redox chain (the ferredoxin-thioredoxin system) leads to reductive light activation of several stromal target enzymes, e.g.fructose-1,6-bisphosphatase, NADP-malate dehydrogenase, phosphoribulokinase, and others (4Scheibe R. Plant Physiol. 1991; 96: 1-3Crossref PubMed Scopus (162) Google Scholar). In contrast, chloroplast G6PDH is inactivated in the light (5Lendzian K. Ziegler H. Planta. 1970; 94: 27-36Crossref PubMed Scopus (43) Google Scholar) or by reductants (6Johnson H.S. Planta. 1972; 106: 273-277Crossref PubMed Scopus (25) Google Scholar, 7Scheibe R. Anderson L.E. Biochim. Biophys. Acta. 1981; 636: 58-64Crossref PubMed Scopus (109) Google Scholar) and is therefore active in the oxidized state. This regulation prevents futile cycling,i.e. simultaneous carbohydrate synthesis in the Calvin cycle and catabolism by the oxidative pentose-phosphate pathway. Thus, in accordance with its physiological role in chloroplasts, G6PDH is active only during the dark phase, when NADPH supply by the photosynthetic electron flow ceases. Recently, we isolated cDNA sequences encoding cytosolic and plastidic G6PDH from potato (8Graeve K. von Schaewen A. Scheibe R. Plant J. 1994; 5: 353-361Crossref PubMed Scopus (109) Google Scholar, 9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar). Both plant isoforms contain six cysteine residues, but none of them at conserved positions. Notably, in the plastidic sequence all cysteines are located within a relatively short amino-terminal stretch of about 100 amino acids (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar) within the NADP binding domain (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Comparison of the deduced amino acid sequences with those of the redox-modulated G6PDH from cyanobacteria (11Scanlan D.J. Newman J. Sebaihia M. Mann N.H. Carr N.G. Plant Mol. Biol. 1992; 19: 877-880Crossref PubMed Scopus (28) Google Scholar, 12Newman J. Karakaya H. Scanlan D.J. Mann N.H. FEMS Microbiol. Lett. 1995; 133: 187-193Crossref PubMed Google Scholar, 13Summers M.L. Meeks J.C. Chu S. Wolf Jr., R.E. Plant Physiol. 1995; 107: 267-268Crossref PubMed Scopus (16) Google Scholar) revealed substantial differences in the primary structures. The cyanobacterial G6PDH sequences contain two conserved cysteines at completely different positions compared with plastidic G6PDH. To locate the cysteine residues involved in redox regulation of the chloroplast enzyme, both recombinant plant isoforms and six mutants of plastidic G6PDH from potato were expressed in Escherichia coli and characterized with respect to inactivation that can be achieved by preincubation with reduced dithiothreitol (DTTred) in vitro (6Johnson H.S. Planta. 1972; 106: 273-277Crossref PubMed Scopus (25) Google Scholar). The data show that only the plastidic enzyme is regulated by redox modification and that two of the six cysteines are involved in this mechanism. The results are discussed based on recent crystallographic data obtained with theLeuconostoc enzyme (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). All biochemicals were of highest purity and purchased from Boehringer (Mannheim, FRG), Sigma (Deisenhofen, FRG), or Biomol (Hamburg, FRG). Restriction endonucleases and DNA-modifying enzymes were obtained through Boehringer (Mannheim, FRG), Life Technologies, Inc. (Eggenheim, FRG), New England Biolabs (Schwalbach, FRG) or MBI Fermentas (St. Leon Rot, FRG). Oligonucleotides for sequencing or site-directed mutagenesis were purchased either from Eurogentec (Seraing, Belgium) or MWG Biotech (Ebersberg, FRG). E. coli strain XL1-Blue served as standard host for cloning in pBluescript II SK (pBSK), or preparation of single-strand DNA in combination with helper phage R408 (Stratagene, Heidelberg, FRG). E. coli strain BL21 (DE3) pLysS was used for overexpression of wild-type and mutant g6pdh cDNA sequences in pET16b (Novagen/AGS, Heidelberg, FRG). In addition, G6PDH-deficient E. coli strain SU294 (14Lee W.T. Levy H.R. Protein Sci. 1992; 1: 329-334Crossref PubMed Scopus (33) Google Scholar) was modified for mutant analysis. To allow for expression of the pET-g6pdhconstructs, SU294 was first transformed with plasmid pGP1-2 (15Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar), a pACYC derivative, carrying the T7 RNA-polymerase gene under control of a heat-inducible lacUV5 promoter. E. coli strains were grown according to standard procedures (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) in media containing the following antibiotics: for XL1-Blue, 10 ॖg/ml tetracycline; for BL21 (DE3) carrying pLysS, 25 ॖg/ml chloramphenicol; for strains transformed with pBSK- or pET-derivatives additionally 200 ॖg/ml ampicillin; and for SU294 carrying pGP1–2, 25 ॖg/ml kanamycin. Oligonucleotides were designed according to conserved regions in the cytosolic and plastidic g6pdh-cDNA sequences from potato. Primers PFL038 and PFL046 have been described previously (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar). Phosphorylation of the oligonucleotides was according to standard procedures (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 舠Sense舡 primer for modification of plastidicg6pdh introducing two 5′-restriction sites (XhoIunderlined, BamHI in bold), corresponding to 63SSNGFPLNA in plastidic G6PDH; 38-mer, 5′-CGGCTCGAG GAT CCC TCA AAT GGG TTT CCA CTT AAT GC-3′. 舠Sense舡 primer for modification of cytosolicg6pdh introducing two 5′-restriction sites (XbaIunderlined, XhoI in bold), corresponding to 1MAASWCI in cytosolic G6PDH; 36-mer, 5′-GGTCTAGA CTCGAG ATG GCG GCA TCA TGG TGT ATT G-3′. Degenerate sequencing primer (舠antisense舡) based on conserved region 233/176VEKPFG in plastidic and cytosolic potato G6PDH, respectively; 17-mer, 5′-CC(A/G) AAN GG(C/T) TT(C/T) TCN AC-3′. Degenerate sequencing primer (舠sense舡) based on conserved region 100/39GDLAKK in plastidic and cytosolic potato G6PDH, respectively; 17-mer, 5′-GGN GA(C/T) (C/T)TN GCN AA(A/G) AA-3′. 舠Antisense舡 primer for mutagenesis of cytosolic g6pdh by PCR, corresponding to79LRSRIRGYLS(149) C RIDKREN (157) C EGEVSEFLQL (cysteines underlined, introduced plastidic g6pdhsequence in bold, sequence numbering as in von Schaewenet al. (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar)); 88-mer, 5′-CAG TTG CAG AAA CTC TGA TAC TTC TCC TTC GCA ATT TTC TCT CTT ATC AAT TCG ACA AGA AAG ATA CCC ACG GAT ACG GCT TCT CAA G-3′. 舠Sense舡 primer for mutagenesis of cytosolicg6pdh by PCR, corresponding to PFL085–2; 88-mer, 5′-C TTG AGA AGC CGT ATC CGT GGG TAT CTT TCT TGT CGA ATT GAT AAG AGA GAA AAT TGC GAA GGA GAA GTA TCA GAG TTT CTG CAA CTG-3′. The following 舠antisense舡 oligonucleotides were used to replace the codons for Cys119, Cys149, Cys157, Cys168, Cys194, and Cys216(sequence numbering as in von Schaewen et al. (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar)) by those coding for serine (codons underlined, exchanged bases inbold). C119S, 5′-CTGAGGCAGAGAATCTTCATAG-3′; C149S, 5′-CAATTCGAGAAGTTAAGG-3′; C157S, 5′-GGCATCGGAATTCTCTC-3′; C168S, 5′-CGAATGATAAAAGGATCTTTCC-3′; C194S, 5′-GAAACCCTAGAACCCTCC-3′; C216S, 5′-GACTTGCAGATCGCACC-3′. All DNA-cloning techniques followed previously described standard methods (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For overexpression of plastidic g6pdh with 10 N-terminal histidine residues (舠His-tag舡), clone pBSK-4.3 carrying the full-length cDNA coding for plastidic G6PDH (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar) was digested with restriction enzymes XhoI and BstEII. The resulting vector fragment (Δ300 bp) was isolated upon agarose-gel eletrophoresis. The same clone served as template for PCR (to introduceXhoI and BamHI restriction sites at the 5′-end) using sense primer PFL070 which corresponds to the deduced mature N terminus of plastidic G6PDH, and internal antisense primer PFL046 which is specific for the plastidic isoform. PCR was conducted as described previously (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar), and the resulting 940-bp product was digested withXhoI and BstEII. The resulting 120-bp fragment was recovered from the gel and ligated to the Δ300-bp vector fragment. From this construct the tailored cDNA fragment was excised with BamHI and inserted into expression vector pET-16b. For overexpression, the final His-tag construct (pET-4.3His) was transformed into E. coli strains BL21 (DE3) pLysS and SU294 pGP1-2. The cloning strategy for overexpression of cytosolic g6pdh was similar to the one described above for the plastidic isoform. Plasmid pBSK-K4 carrying the full-length cDNA for cytosolic G6PDH (8Graeve K. von Schaewen A. Scheibe R. Plant J. 1994; 5: 353-361Crossref PubMed Scopus (109) Google Scholar) was digested to completion with XbaI and partially with EcoRI. The resulting vector fragment (Δ400 bp) was gel-purified. From the same clone PCR was conducted with primers PFL071 (to introduceXbaI and XhoI restriction sites at the 5′-end) and PFL038. The 650-bp product was digested with XbaI andEcoRI, the resulting 380-bp fragment was recovered from the gel and ligated to the Δ400-bp vector fragment. From this construct the modified cDNA fragment was excised with XhoI and inserted into expression vector pET-16b. For overexpression, the final His-tag construct (pET-K4His) was transformed into E. BL21 (DE3) pLysS and SU294 pGP1-2. mutagenesis of the cysteine codons was conducted with the in vitro mutagenesis FRG) based on the J. 1985; Scopus Google Scholar, PubMed Scopus Google Scholar). To a construct that allow for both of single-strand DNA and plasmid pBSK-4.3 was modified as The region of was by and and the resulting vector fragment bp) was subsequently ligated to the fragment of This construct was digested with BamHI and ligated to the fragment of to allow from the T7 in pET-16b. The resulting construct was used for single-strand DNA for site-directed mutagenesis. From this expression were for G6PDH site-directed the the base were therefore into The plastidic sequence Cys149 and Cys157 was into by PCR using oligonucleotides and the mutagenesis (Stratagene, Heidelberg, FRG). The of the DNA was by sequence each cloning based on the chain Fritsch E.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) were conducted with plasmid DNA plasmid FRG) using the plasmid sequencing FRG). Degenerate primers and were used at 10 of plasmid For the synthesis of recombinant E. were grown at in containing the of was to final at for the were by 10 in of the 100 and to two in To the was for on in a the was used for G6PDH of protein was according to Biochem. PubMed Scopus Google Scholar) using as For gel proteins were in 1970; PubMed Scopus Google Scholar) and with FRG) served as a of recombinant His-tag proteins on followed for the of As to was with G6PDH activity was at in a FRG). G6PDH were as described previously (8Graeve K. von Schaewen A. Scheibe R. Plant J. 1994; 5: 353-361Crossref PubMed Scopus (109) Google Scholar). inactivation of G6PDH was in the of of either or All were at under containing were with and to The standard inactivation the and in a of and was for 10 at to enzyme To the of reductive preincubation was in a and were different For of the reduced enzyme, were with the same of or in 100 a or DTTred, to G6PDH The corresponding were with To the of thioredoxin on reductive were with and of recombinant thioredoxin recombinant plastidic G6PDH in of plastidic G6PDH (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar) upon with the amino acid sequence from W.T. C. Levy H.R. J. Biol. 1991; Full Text PDF PubMed Google Scholar) was using G. J. Mol. PubMed Scopus Google Scholar), M. A. 1991; PubMed Scopus Google Scholar), and Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). of the (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) were obtained from the Protein expression of plastidic G6PDH as a protein led to enzyme activity (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar), the two plant G6PDH isoforms were overexpressed as amino-terminal His-tag proteins in E. coli strain Both exhibited G6PDH activity in that was at for several addition of enzyme activity was The of plastidic G6PDH, in of the cytosolic isoenzyme to the recombinant enzymes by their amino-terminal under native not but in inactivation of both This was for and on Heidelberg, FRG), and with Heidelberg, FRG). the of different in the standard G6PDH activity and addition of activity not we that inactivation of the G6PDH isoforms in and is to a of the The of the recombinant enzymes were therefore in different G6PDH were least of the activity in and are in as of the of different on the activity of both G6PDH was by reduced was only for the plastidic isoform. The activity of the cytosolic enzyme in was not influenced The of inactivation of the plastidic with either or was on and could be completely reversed by addition of in for which that the enzyme was not inactivation of recombinant plastidic G6PDH by and subsequent of the reduced enzyme with with was followed by with 100 were in using the standard is the of two In chloroplasts, are known to redox modification of stromal target recombinant thioredoxin was during preincubation with inactivation of the plastidic enzyme was This was specific for thioredoxin m and not with The activity of the cytosolic enzyme was not influenced by either thioredoxin not Comparison of the cysteine positions in and cyanobacterial G6PDH sequences not which of the six residues in the mature plastidic enzyme be involved in redox regulation each of the six cysteine codons in the plastidic cDNA sequence from potato was exchanged for serine by In addition, to in the of the mutagenesis the G6PDH-deficient E. coli strain SU294 (14Lee W.T. Levy H.R. Protein Sci. 1992; 1: 329-334Crossref PubMed Scopus (33) Google Scholar) was for the expression of wild-type and mutant in expression of the recombinant plant isoforms in SU294 for G6PDH activity In control from either or the recombinant G6PDH activity was Two of the six mutant proteins and compared with the in the standard In the of DTTred, G6PDH activity of the to of the control In contrast, the activity of and was influenced by the mutants or The enzyme were compared at different from to 100 of the recombinant wild-type and mutant enzymes C168S, C194S, and in G6PDH activity under but not under In contrast, on the activity of mutants and C157S, that the abolished redox In all were at To the of the oxidized and reduced m of wild-type and mutant enzymes were from The recombinant wild-type enzyme compared with the native enzyme from R. A. K. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). The m of the oxidized wild-type enzyme and is upon reduction. mutant C119S, C168S, C194S, and C216S, show m upon reduction. In contrast, the m of both the oxidized and reduced of mutants and and within the of the for the reduced wild-type enzyme. with data obtained with the native chloroplast enzyme from R. A. K. Arch. Biochem. Biophys. PubMed Scopus Google Scholar), m for the NADP was with either the recombinant wild-type or the mutant proteins not m for of oxidized and reduced plastidic G6PDH Scheibe et al. Scheibe et al. were from a one in From Scheibe et al. R. A. K. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). in a were from a one in of the of plastidic G6PDH was based on a of the deduced amino acid sequence (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar) with the crystallographic of the enzyme (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). the resulting of plastidic potato G6PDH as a of that amino acids Cys149 and Cys157 are the only for The two cysteine residues to be located in on the of the and be for with The the is which is to the for To the of the containing the two cysteines on the activity of the cytosolic region Cys149 and Cys157 in plastidic G6PDH was introduced into the cytosolic by PCR, the sequence amino acid von Schaewen et al. (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google this modification not the activity of the cytosolic enzyme in the or of not mutagenesis is a to of recombinant this the responsible cysteine residues in NADP-malate dehydrogenase E. M. P. C. P. M. J. Biol. 1994; Full Text PDF PubMed Google Scholar) and J. M. S. J. A. J. Lett. PubMed Scopus Google Scholar), two target enzymes of the ferredoxin-thioredoxin of have been to we show which cysteines are involved in redox regulation of plastidic G6PDH. This protein a in to all known enzymes, plastidic G6PDH is inactivated by (5Lendzian K. Ziegler H. Planta. 1970; 94: 27-36Crossref PubMed Scopus (43) Google Scholar, 7Scheibe R. Anderson L.E. Biochim. Biophys. Acta. 1981; 636: 58-64Crossref PubMed Scopus (109) Google Scholar). Recently, we the cDNA sequences coding for cytosolic and plastidic G6PDH from potato (8Graeve K. von Schaewen A. Scheibe R. Plant J. 1994; 5: 353-361Crossref PubMed Scopus (109) Google Scholar, 9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar). Both isoforms contain six cysteine residues in their deduced amino acid sequences All cysteine residues in cytosolic and plastidic G6PDH proteins different and one are located at conserved positions within the and A. von were with both recombinant G6PDH from potato. could show that only plastidic G6PDH is inactivated upon in regulation of the recombinant cytosolic as described for the enzyme L.E. Plant Physiol. PubMed Google Scholar, L.E. Plant Physiol. PubMed Google Scholar, L.E. Lett. PubMed Scopus Google Scholar, Anderson L.E. Plant Physiol. PubMed Google Scholar), could not be of recombinant plastidic G6PDH is fully reversible by and is not to of The of thioredoxin on the of reductive inactivation of chloroplast G6PDH been described R. Anderson L.E. Biochim. Biophys. Acta. 1981; 636: 58-64Crossref PubMed Scopus (109) Google Scholar), and was for the recombinant enzyme not As been for the enzyme in R. A. K. Arch. Biochem. Biophys. PubMed Scopus Google Scholar), the m for was from in the oxidized to in the reduced with the the m for the NADP was not with the recombinant plastidic enzyme. The results obtained that recombinant plastidic G6PDH expressed in E. coli its native This was the for the of redox regulation of plastidic G6PDH with respect to the of cysteine residues by site-directed mutagenesis. led to enzyme the of the recombinant wild-type and the mutant plastidic G6PDH enzymes were characterized in of a G6PDH-deficient E. coli of two of the six cysteines by abolished the of the enzyme. The for the reduced and oxidized enzyme were and the m were to those of the reduced wild-type enzyme. As the The by dithiothreitol that the two mutant proteins and the wild-type enzyme in the state. results are with both residues in a in active plastidic G6PDH. on the crystallographic data obtained with the enzyme (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), of the plastidic potato sequence revealed that only the two cysteines and be to a The two cysteine residues in the amino-terminal domain of the enzyme which the The m for the were by the A similar was obtained upon of a conserved in the binding domain of the enzyme which of the (10Rowland P. Basak A.K. Gover S. Levy H.R. Adams M.J. Structure. 1994; 2: 1073-1087Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). binding in the domain of the we that of the results in and prevents either binding of the or of the and NADP binding of the of the plastidic the two cysteines into cytosolic G6PDH not the activity of the enzyme under or not This the differences that exist the two plant the recombinant proteins only their G6PDH in plant (9von Schaewen A. Langenkämper G. Graeve K. Wenderoth I. Scheibe R. Plant Physiol. 1995; 109: 1327-1335Crossref PubMed Scopus (75) Google Scholar). In the data that the two cysteines in the plastidic potato sequence are but not for redox regulation of G6PDH from are to H. Levy for the G6PDH-deficient E. for recombinant thioredoxin m and for with of plastidic G6PDH, for and for of the