The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD+ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering. TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD+ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering. Tyrosine serves as a precursor for the synthesis of proteins and secondary metabolites such as quinones (1Meganathan R. FEMS Microbiol. Lett. 2001; 203: 131-139Crossref PubMed Google Scholar, 2Meganathan R. Vitam. Horm. 2001; 61: 173-218Crossref PubMed Google Scholar, 3Rinaldi A.C. Porcu C.M. Oliva S. Curreli N. Rescigno A. Sollai F. Rinaldi A. Finazzi-Agro A. Sanjust E. Eur. J. Biochem. 1998; 251: 91-97Crossref PubMed Scopus (20) Google Scholar), alkaloids (4Memelink J. Nat. Biotechnol. 2004; 22: 1526-1527Crossref PubMed Scopus (13) Google Scholar), flavonoids (5Kaneko T. Baba N. Matsuo M. Chem. Biol. Interact. 2003; 142: 239-254Crossref PubMed Scopus (65) Google Scholar), and phenolic compounds (5Kaneko T. Baba N. Matsuo M. Chem. Biol. Interact. 2003; 142: 239-254Crossref PubMed Scopus (65) Google Scholar, 6Mobley E.M. Kunkel B.N. Keith B. Gene (Amst.). 1999; 240: 115-123Crossref PubMed Scopus (73) Google Scholar). In prokaryotes and plants, these compounds are important for viability and normal development (7Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (482) Google Scholar). The TyrA protein family consists of dehydrogenase homologues that are dedicated to the biosynthesis of l-tyrosine. These enzymes participate in two independent metabolic branches that result in the conversion of prephenate to l-tyrosine, namely the arogenate route and the 4-hydroxyphenylpyruvate (HPP) 3The abbreviations used are: HPP, 4-hydroxyphenyl pyruvate; PD, prephenate dehydrogenase; AD, arogenate dehydrogenase; HPpropionate, hydroxyphenylpropionate; CM-PD, chorismate mutase-prephenate dehydrogenase. routes. Although both of these pathways utilize a common precursor and converge to produce a common end-product, they differ in the sequential order of enzymatic steps. Through the HPP route, prephenate is first decarboxylated by prephenate dehydrogenase (PD) to yield HPP, which is subsequently transaminated to l-tyrosine via a TyrB homologue (8Fazel A.M. Bowen J.R. Jensen R.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1270-1273Crossref PubMed Scopus (26) Google Scholar). Alternatively, through the arogenate route, prephenate is first transaminated to l-arogenate by prephenate aminotransferase and then decarboxylated by arogenate dehydrogenase (AD) to yield l-tyrosine (9Bonner C. Jensen R. Methods Enzymol. 1987; 142: 488-494Crossref PubMed Scopus (18) Google Scholar, 10Bonner C. Jensen R. Methods Enzymol. 1987; 142: 479-487Crossref PubMed Scopus (22) Google Scholar, 11Fazel A.M. Jensen R.A. J. Bacteriol. 1979; 138: 805-815Crossref PubMed Google Scholar) (see Fig. 1A). There are three classes of TyrA enzymes that catalyze the oxidative decarboxylation reactions in these two pathways. The enzymes are distinguished by the affinity for cyclohexadienyl substrates. PD and AD accept prephenate or l-arogenate, respectively, whereas the cyclohexadienyl dehydrogenases can catalyze the reaction using either substrate (12Song J. Bonner C.A. Wolinsky M. Jensen R.A. BMC Biol. 2005; 3: 13Crossref PubMed Scopus (24) Google Scholar). To ensure efficient metabolite distribution of the pathway intermediates, TyrA enzymes are highly regulated by various control mechanisms, including feedback inhibition, and genetic regulation by the Tyr operon (13Bonner C.A. Disz T. Hwang K. Song J. Vonstein V. Overbeek R. Jensen R.A. Microbiol. Mol. Biol. Rev. 2008; 72 (table of contents): 13-53Crossref PubMed Scopus (28) Google Scholar, 14Bonner C.A. Jensen R.A. Gander J.E. Keyhani N.O. Biochem. J. 2004; 382: 279-291Crossref PubMed Scopus (22) Google Scholar, 15Cobbett C.S. Delbridge M.L. J. Bacteriol. 1987; 169: 2500-2506Crossref PubMed Google Scholar, 16Xia T. Zhao G. Jensen R.A. J. Mol. Evol. 1993; 36: 107-120Crossref PubMed Scopus (9) Google Scholar). In some cases, l-tyrosine competes directly with substrate, be it prephenate or l-arogenate for the active site of arogenate or cyclohexadienyl dehydrogenases (14Bonner C.A. Jensen R.A. Gander J.E. Keyhani N.O. Biochem. J. 2004; 382: 279-291Crossref PubMed Scopus (22) Google Scholar, 17Rippert P. Matringe M. Eur. J. Biochem. 2002; 269: 4753-4761Crossref PubMed Scopus (62) Google Scholar, 18Xie G. Bonner C.A. Jensen R.A. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2000; 125: 65-83PubMed Google Scholar, 19Bonvin J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar). The product HPP can also serve as an efficient competitive inhibitor with respect to prephenate (20Jensen R.A. Stenmark S.L. J. Mol. Evol. 1975; 4: 249-259Crossref Scopus (19) Google Scholar). Additionally, at the protein level PDs are only shown to be regulated at distinct allosteric sites or domains to modulate their activity. For example, the results of kinetic studies on the bifunctional Escherichia coli chorismate mutase-prephenate dehydrogenase (CM-PD) have indicated that this enzyme likely possesses a distinct allosteric site for binding tyrosine (21Turnbull J. Morrison J.F. Cleland W.W. Biochemistry. 1991; 30: 7783-7788Crossref PubMed Scopus (18) Google Scholar). In contrast, the Bacillus subtilis PD is the only enzyme reported to be competitively by HPP and l-tyrosine is also by and (12Song J. Bonner C.A. Wolinsky M. Jensen R.A. BMC Biol. 2005; 3: 13Crossref PubMed Scopus (24) Google Scholar, Jensen R.A. J. Biol. Chem. PubMed Google Scholar). control is to through a of the B. subtilis PD B. Curr. Opin. Biol. 2001; PubMed Scopus Google Scholar). of PD E. coli have a for understanding the molecular mechanism of the TyrA The E. coli reaction a kinetic mechanism with catalysis as the P. Morrison J.F. PubMed Scopus (20) Google Scholar). Additionally, studies of the of the kinetic and that a prephenate to NAD+ by the 4-hydroxyl of prephenate, whereas a is for binding prephenate to the J. Cleland W.W. Morrison J.F. Biochemistry. 1991; 30: PubMed Scopus (22) Google Scholar). The residues and have through studies to these two D. Turnbull J.L. Biochemistry. 1999; PubMed Scopus (26) Google Scholar, D. Turnbull J.L. Biochemistry. 1998; PubMed Scopus Google Scholar). of the of protein and site-directed in the of a of inhibitory substrate the that binds prephenate through the D. Turnbull J.L. Biochemistry. 1999; PubMed Scopus (26) Google Scholar). The structures of AD and PD Aquifex aeolicus in with have reported by P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar) and by our Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar), respectively. of these structures have structural on the and The structure A. aeolicus PD also to the identification of active site residues that a in enzyme most which we catalysis by the catalytic and the of NAD+ their efficient can of the prephenate only in the active site. For example, of the AD structure by P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar) to in E. coli and Arg-250 in A. the active site to a in prephenate the of prephenate and TyrA proteins are as are the of the enzymes with l-tyrosine. To further the of residues in ligand and we have studies of A. aeolicus PD with NAD+ and prephenate, with NAD+ and a product and with NAD+ and l-tyrosine. this provides the first that l-tyrosine binds to the active site of a prephenate dehydrogenase. have the of and Arg-250 through the kinetic of site-directed and structural of the To the of active site residues in substrate structural of AD and PD also The provides a for understanding the mechanism of substrate selectivity the classes of TyrA enzymes and A. aeolicus PD can accept prephenate as substrate and l-tyrosine as a competitive and as Morrison J.F. Biochem. Google Scholar), NAD+ whereas HPP and The of HPP as in B. G. S. M. J. Biol. Chem. PubMed Google Scholar). of molecular and or used for site-directed residues of the A. aeolicus PD protein J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar). using the site-directed the for the and the used for mutagenesis. by used to active site the site for to in mutant the site for to in mutant in a Protein and of A. aeolicus PD and as Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar) with the mutant by by and the of the enzyme on PDs directly to on a of and for of A. aeolicus PD in the of NAD+ and prephenate in a reaction and at as J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar). the reaction at for and then the reaction by the of The of at The and for by to the for the of HPP, HPpropionate, or l-tyrosine the by to the for competitive These by prephenate at to the the of and HPP to and l-tyrosine to as a of l-tyrosine as by J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar). kinetic to the by using the of Cleland W.W. Methods Enzymol. 1979; PubMed Scopus Google Scholar) or in as a of prephenate used to of the prephenate the as by J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar). Protein using the Protein with as a of determined with a at using the The consists of and at and by the protein a of with NAD+ and of or prephenate, or l-tyrosine. and at a of in a on at a at the The and with the of Methods Enzymol. PubMed Scopus Google Scholar). The structures with the structure Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar) to the of the of and P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar), to the and with the A. 1991; PubMed Scopus Google Scholar). The of the with and subsequently with of In the of and structure and with respectively, used for the and of the P. K. Biol. 2004; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Methods Enzymol. 2003; PubMed Scopus Google Scholar). of PD to of and not NAD+ and to the in in the the of and for tyrosine, HPpropionate, and HPP the Biol. PubMed Scopus Google Scholar, The Scholar). the crystal structure with The of this to active site residues that are directly in the catalytic and of PD and to the mechanism that to substrate selectivity enzymes in the TyrA in the of NAD+ and either prephenate, HPpropionate, or l-tyrosine. studies residues and which are important for either the reaction or for ligand and of and of and and These belong to and their structures determined by molecular as by the for the structures of with and of the residues are in the The structure of and to and respectively. and are in and of in in in to the in to the in a in of residues of or in a Although for the of these the residues in the to or for the residues and residues for residues and for residues and for residues and for crystal residues and in and in and in and and in for the crystal residues and in and residues in and in and and in D. of in these of the to the normal and of and studies with NAD+ and of or l-tyrosine, respectively. the NAD+ and The of in the protein crystal determined at The of HPP and of prephenate and in the that prephenate to HPP the Fig. the structure of ligand used in structure of ligand in the the of these the to the of of the product or product is In contrast, both in the a of NAD+ or For example, for of the a of and of product this as a whereas the only of product this as the is that in the crystal the binding of HPP to both binding sites the structure The of NAD+ in the active site that NAD+ binds first by HPP to be the active site. is highly this is by the that we that HPP, in the of the active site. is also likely that NAD+ binding is for The of and of product in the is that substrate binding and product are which is with kinetic for arogenate dehydrogenase in which arogenate binds first (14Bonner C.A. Jensen R.A. Gander J.E. Keyhani N.O. Biochem. J. 2004; 382: 279-291Crossref PubMed Scopus (22) Google Scholar). this finding the kinetic mechanism the of and and product studies of PD and AD a of including E. coli and P. Matringe M. Eur. J. Biochem. 2002; 269: 4753-4761Crossref PubMed Scopus (62) Google Scholar, 19Bonvin J. Aponte R.A. Marcantonio M. Singh S. Christendat D. Turnbull J.L. Protein Sci. 2006; 15: 1417-1432Crossref PubMed Scopus (15) Google Scholar, P. Morrison J.F. PubMed Scopus (20) Google Scholar, P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar). of the crystal structure of PD indicated that substrate binding a on the The protein is in a with only NAD+ bound and the binding of HPP or a product the two by and is on the the is in the that this the binding of product in this to the the active site residues in the are in their to with the The binding of the product structural the PD and of structure with the structure indicated that the structures of the are to in the in that the active site The most in the residues and a which the or the of the active site. of this that this substrate The of HPP, HPpropionate, or l-tyrosine in the active site that this is to and the of the product or product and In this a that is to participate in the mechanism through of an with and we have on our structural analyses, that binding through this by substrate to the active site Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar). of the that the with Arg-250 is with substrate the of the product this is secondary structures that have ligand binding and important His-217 and are these respect to the the of Arg-250 in the is to the active site and to the of the of the A. PD of the structures of A. aeolicus PD with of the three HPP, HPpropionate, and l-tyrosine, to residues in substrate binding and and in the regulation of enzyme by inhibitor, l-tyrosine. of the have a common they in the of the at the These at the the binding of the to A. aeolicus PD HPP, the as a competitive inhibitor with respect to prephenate for the reaction by not The of HPP the of is to the for prephenate of HPP is a and the with prephenate on the structural these two we that the HPP and active site residues of PD with by prephenate in the of and of l-tyrosine also to the for competitive not with a of for of for and dehydrogenases at and using a of NAD+ for the prephenate the substrate NAD+ at NAD+ the substrate, prephenate at and at and prephenate to using of for NAD+ not not using a of NAD+ for the prephenate the substrate NAD+ at NAD+ the substrate, prephenate at and NAD+ at and prephenate to using of for NAD+ not not in a of the of the active site of A. aeolicus PD is with our with prephenate Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar). The active site to the of which is for efficient prephenate to the of the both of the to the prephenate binding a of the binding is at the Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar). The active site can be three on and with the product a of residues and a of residues and and an of residues and and the of these three in the active site the with the of the The of the ligand is in the whereas the participates in with residues in the of His-147 in the the of the HPpropionate, and and active site residues are The with a highly catalytic which is of a Ser-126 and and a highly and Additionally, of the ligand is also the of the of These are with kinetic E. coli CM-PD, which to A. aeolicus as a key catalytic group. for the E. coli of revealed that a most likely His-147 with a of is for catalysis not also on the A. aeolicus PD mutant to in catalysis The mutant is binds prephenate with affinity to the as determined by kinetic and by of the of by prephenate not These further the catalytic of His-147 in the of prephenate to prephenate to of Ser-126 in the of Ser-126 further by and kinetic Ser-126 an with the of NAD+ and and a directly in catalysis by these in a In this also to the binding of the and prephenate in the active site. both a in a in the for prephenate, and a in for tyrosine, whereas the binding of NAD+ is with the of Ser-126 in the with prephenate and A. aeolicus PD in a of in the is a highly that participates in the with the this is only in the of the enzyme with NAD+ and the is shown to the and the of the ligand and that serve two be in the binding with the ligand participate in the of His-147 in the catalytic of Arg-250 in the of the is HPP, HPpropionate, and l-tyrosine of the structure in with of the three revealed that this directly with the of Arg-250 and is in of this by an In these Arg-250 is with In the of a Arg-250 is by the of that it an important binding of the mutant a in the for prephenate and a in for tyrosine to the enzyme in the affinity for NAD+ or these are in with Arg-250 it is not for the binding of the via the group. Arg-250 not prephenate or tyrosine is that or active site residues are also important for ligand of His-217 in the most important the of the are: HPP a in to the l-tyrosine possesses an and is a at the The structure of the that the on this can with the of and is also a which is by a of of the and the of HPP and The of His-217 is also to the of These by both and of His-217 that it is in the To further the of and and of His-217 to either or produce mutant enzymes that are not by tyrosine also have in their kinetic for the The for prephenate by and for the and respectively, the of His-217 in prephenate selectivity these also with to in that this with the of the substrate in prephenate in a in kinetic for both and can also be in to structural on the active site to of the of His-217 with the and that and participate in a with and this is the ligand is not and also determined that the binding of NAD+ and the for NAD+ by for both His-217 of the on in E. coli effect on both the for prephenate, a and the a with effect on NAD+ binding D. Turnbull J.L. Biochemistry. 1998; PubMed Scopus Google Scholar). this E. coli mutant is also to tyrosine, at of tyrosine, and not The of tyrosine to the E. coli mutant provides further for the of this in tyrosine E. coli with A. aeolicus PD in a that the of these two active sites is not such in the active site residues PDs the of in kinetic the and A. aeolicus PD and E. coli The of His-217 is further by the structure of with the and as such participate in the at this of HPP and in the active site that the are not the is to His-217 for the of HPP to E. coli PD is that for HPpropionate, D. Turnbull J.L. Biochemistry. 1999; PubMed Scopus (26) Google Scholar). These are in with our that or residues in the of His-217 are as a for the binding of to PD active site. the can with affinity of of their The structure of in with tyrosine revealed a of with the of tyrosine with with the of HPP and In this the of l-tyrosine is in the with that of the of a the of l-tyrosine is His-217 and the by and is with the of and of the in the active site of PD revealed that the of l-tyrosine His-217 by with the of HPP, and the the of the and His-217 is to Tyrosine of the three in the A. aeolicus PD active site revealed that the a on the of a HPP, or an in with the kinetic studies of the His-217 to that the His-217 is important for tyrosine further that the of His-217 and with active site residues the of that can to the active site of on the at both and for we that the of this is in the is with our for the and in which the of HPP is the of His-217 and the of l-tyrosine is the and is with the of is likely that this of His-217 produce a which the the of and the of the bound Tyrosine studies to the of His-217 and residues in the binding of l-tyrosine to shown to have an inhibitory effect on both enzyme and on a inhibitory effect on the of is by l-tyrosine the the is the of the bound tyrosine and The most interesting finding is the inhibitory effect of l-tyrosine by mutating His-217 to either an or an the binding of prephenate is also by the and which that mutating His-217 result in structural of the active which a effect on tyrosine In our results mutational of the E. coli enzyme that the to in A. also feedback by l-tyrosine, and for prephenate is that His-217 the effect the which is responsible for the the of tyrosine and the of of AD and of an AD structure structural and TyrA dehydrogenases for understanding of the molecular mechanism responsible for substrate binding in the TyrA family of To this we the and structures with that of and the structure of with the AD structure P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar) The and have an of The structures PD and AD are their secondary structures have of the and and and P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar, Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar) the active site of AD is and to that of is to the that and the which the and of the respectively, are the active site P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar, Singh S. R. Turnbull J.L. Christendat D. J. Biol. Chem. 2006; PubMed Scopus (19) Google Scholar). of the PD and AD active sites that important residues are and For example, the catalytic His-147 in PD, and the important binding Arg-250 in PD, are in both the residues that are shown to to the of prephenate are also common active site residues and to the A. the of the of in the binding of this also the of The of important residues that l-arogenate to the AD active site in a to that of prephenate in the HPP the structure the AD active site this on the we that it is for l-arogenate to with and which to and Arg-250 in with P. R. M. P. R. J.L. Matringe M. 2006; PubMed Scopus Google Scholar) have that this is the active site to a in substrate these in the active are is a to the HPP in the AD active site and results the of and residues and His-217 in and the of and in the AD structure These residues the binding for AD is a to His-217 and is not by finding is in with our of His-217 in selectivity for tyrosine and the of active site residues of proteins in the TyrA residues that are in ligand and structural with structures in the revealed that the catalytic is proteins in the TyrA The that is in binding the through the is also The most is that the of His-217 l-tyrosine and we that it the binding of to the PD active site. the of His-217 l-tyrosine to as a competitive inhibitor with respect to of the at the inhibitory effect of l-tyrosine on A. aeolicus PD activity. novel finding on metabolic of pathways in is likely that tyrosine as a feedback by PD at The of the His-217 produce a active PD, which result in the unregulated of HPP and subsequently an in is known to be an precursor of the biosynthesis of is the of secondary and in is used as a precursor for the synthesis of including and 2000; PubMed Scopus Google Scholar). and for their of the

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.