THE JOURNAL 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 263. No. 24, Issue of August 25, pp. 11780-11785,1988 Printed in U.S.A.

Primary Structure of the Light-dependent Regulatory Site of Corn NADP-Malate Dehydrogenase*

(Received for publication, January 15,1988)

Paulette Decottignies$& Jean-Marie Schmitterll, Myroslawa Miginiac-Maslow$, Pierre Le Marechala, Jean-Pierre JacquotS, and Pierre Gadal$ From the SLaboratoire de Physiologie Vegetale Moleculaire, Unite Associee 1128 Centre National de la Recherche Scientifique, Uniuersite Paris-Sud, 91405 Orsav Cedex. France and the TLaboratoire de Biochimie, Ecole Polytechnique, 91 128 Palaiseau Cedex, France

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been pho- toactivated in vitro in a chloroplast system reconsti- tuted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [’“C] iodoacetate incorporation). “C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In ad- dition, the analysis of the tryptic digest of light-acti- vated [‘*C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys” and Cys“, indicating that these 2 residues play a major role in the light activation mechanism. More- over, an activation model, in which photoreduced thio- redoxin was replaced by the dithiol reductant dithio- threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorpo- rate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denatur- ating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys” and Cys“.

NADP-malate dehydrogenase catalyzes the reduction of oxaloacetate to malate in leaves of higher plants. In uiuo, this chloroplastic enzyme requires activation by light, via a specific system called the ferredoxin-thioredoxin system, in order to be catalytically active (1, 2). The ferredoxin-thioredoxin sys- tem is composed of ferredoxin, ferredoxin-thioredoxin reduc- tase, and thioredoxin rn. In vitro, the activation of NADP- MDH’ can be achieved through the photosynthetic electron

*This work received financial support from the Ministbre de 1’ Education Nationale (Action de Recherche Intigrie Chimie-Biolo- gie). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. The abbreviations used are: NADP-MDH, NADP-malate dehy-

drogenase; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatogra- phy.

transport by illumination in a reconstituted system which comprises thylakoids and all the purified proteins listed above (3-5). Activation can also be obtained with thioredoxin in the presence of a reductant such as dithiothreitol (DTT) (5,6). It has been suggested that oxidoreduction reactions are involved in the light regulation process through interconversion be- tween an inactive disulfide form and an active dithiol form. Jacquot et al. (7) have shown that the appearance of 4 thiol groups/mol of enzyme accompanies the activation of NADP- MDH by thioredoxin and DTT. The enzyme being dimeric, with two apparently identical subunits (8), this observation suggested the reduction of one disulfide bridge/subunit. It is likely that light proceeds by the same way; a recent work provides arguments on behalf of this hypothesis (9).

However, until now no work was ever devoted to the study of purified light-activated NADP-MDH. In addition, while spinach ferredoxin and thioredoxin have been sequenced (10, 11) there is no structural information available about the catalytic or regulatory sites of NADP-MDH and little is known about the structure of light-activated enzymes. Re- cently, the sequence of some tryptic peptides of pea NADP- MDH (12) and spinach fructose-1,6-bisphosphatase (13) have been reported.

In this report, we present evidence that corn NADP-MDH is regulated by light via the reduction of a disulfide bridge/ subunit, and we describe the primary structure of the regula- tory site and its location in the amino acid sequence of the protein.

EXPERIMENTAL PROCEDURES

Materials Plant Material-Corn plants (Zea mays var W64 A x W182 E) and

pea plants were grown as described previously (14). Spinach leaves were purchased from a local market.

Reagents-Iodo[l-14C]acetic acid (2 MBqlpmol) was obtained from Centre #Etudes Atomiques (Saclay, France) and diluted with unla- beled sodium iodoacetate (Fluka) as needed. Reagents used for the amino acid sequence were supplied by Applied Biosystems Inc. (Foster City, CA).

Methods Enzyme Purification-Corn leaf NADP-MDH was purified as pre-

viously described (8), except that a fast protein liquid chromatography step using an anion exchange column (mono Q, Pharmacia LKB Biotechnology Inc.) was added. Ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin m were purified from spinach leaves as described in Refs. 15-17, respectively, with one modification, ferre- doxin-thioredoxin reductase was stored without 2-mercaptoethanol. Pea thylakoids were prepared as described in Ref. 18.

NADP-MDH Activity Determination-NADP-MDH activity was measured spectrophotometrically as described previously (14). One

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Regulatory Site of NADP-MDH 11785

used, hydrophobic interactions can occur between the regu- latory site of corn NADP-MDH and the active site of spinach thioredoxin. Since this active site sequence is strictly con- served (except for the first proline residue) in all the thiore- doxins sequenced so far (25-28), corn thioredoxin may contain the same one, and such interactions can be postulated in the homologous system between corn NADP-MDH and thiore- doxin. This hypothesis is consistent with the finding that organic solvent and high ionic strength enhance the activation of corn NADP-MDH by thioredoxin (29). Another interesting feature of the amino-terminal sequence of NADP-MDH is the presence of many charged and hydrophilic amino acids outside the bridge (3 Glu, 2 Asp, 4 Lys, 2 Ser, 4 Thr). It is to be noted that the homology between the N-terminal sequences of pea (12) and corn NADP-MDH is equal to 53% (calculated on 26 amino acids). The sequence inside the S-S bridge is very homologous, except that the first phenylalanine residue is replaced by a tyrosine in pea enzyme.

Our data show that the sulfhydryls that are involved in light activation are involved also in nonphysiological activa- tions, by DTT alone or by DTT and thioredoxin. This finding confirms, a t a molecular level, that the use of such reductants provides valid models to study the activation process.

NADP-MDH is believed to be composed of two identical subunits (8). In accordance with this, during Edman degra- dation a single amino acid was released at each cycle; however, since half or more of the protein did not undergo any cleavage, we cannot assert that the two subunit sequences are totally identical.

Recently, Porter and Hartman (30) showed that spinach phosphoribulokinase, a light-modulated enzyme, contained an active-site sulfhydryl group which was involved in the thio- redoxin-mediated regulation of the enzyme. The tryptic pep- tide containing this residue was sequenced and observed to be derived from the amino terminus of the kinase. It is of interest to note that this cysteinyl residue occupies position 16 in the primary structure to compare with positions 10 and 15 in NADP-MDH. More recently, Porter et al. (31) identified CysS5 as the second cysteinyl residue of the regulatory disul- fide. No other sequence homology between the amino-termi- nal regions of phosphoribulokinase (32) and NADP-MDH can be noticed. Some other chloroplastic enzymes are regu- lated by light via the ferredoxin-thioredoxin system including fructose-1,6-bisphosphatase, sedoheptulose-l,7-bisphospha- tase, NADP-glyceraldehyde-3-phosphate dehydrogenase, and the “coupling factor” (CFl-ATPase). There is no available information about the redox regulatory site of these enzymes. The finding that the thioredoxin-binding domain is located in the amino terminus of both phosphoribulokinase and NADP-MDH should lead to examining this region in all the thioredoxin-modulated enzymes.

In the case of NADP-MDH, it is not demonstrated whether sulfhydryls generated by light activation are required for catalytic activity. Until now, N-bromoacetylethanolamine phosphate, the reagent used by Porter and Hartman for phosphoribulokinase (30), failed to act as a competitive inhib- itor of NADP-MDH activity? Ashton (33) suggested, from experiments based upon Procion Red binding, that the regu- latory dithiol would be distinct from the active site of the enzyme. We are currently investigating this point with more specific reagents.

P. Decottignies, manuscript in preparation.

Acknowledgments-We wish to thank Professor S. Blanquet for his kind interest and encouragement during the work, and Dr. C. Critin for helpful discussions.

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