local changes in the catalytic site of mammalian histidine ...local changes in the catalytic site of...

Local changes in the catalytic site of mammalian histidinedecarboxylase can affect its global conformation and stability

Carlos Rodrıguez-Caso1, Daniel Rodrıguez-Agudo1, Aurelio A. Moya-Garcıa1, Ignacio Fajardo1,Miguel Angel Medina1, Vinod Subramaniam2,* and Francisca Sanchez-Jimenez1

1Department of Molecular Biology and Biochemistry, Faculty of Sciences, Malaga, Spain; 2Max Planck Institute for Biophysical

Chemistry, Goettingen, Germany

Mature, active mammalian histidine decarboxylase is a di-meric enzyme of carboxy-truncated monomers (� 53 kDa).By using a biocomputational approach, we have generated athree-dimensional model of a recombinant 1/512 fragmentof the rat enzyme, which shows kinetic constants similar tothose of the mature enzyme purified from rodent tissues.This model, together with previous spectroscopic data, al-lowed us to postulate that the occupation of the catalyticcenter by the natural substrate, or by substrate-analogs,would induce remarkable changes in the conformation ofthe intact holoenzyme. To investigate the proposed con-formational changes during catalysis, we have carried out

electrophoretic, chromatographic and spectroscopic analy-ses of purified recombinant rat 1/512 histidine decarboxylasein the presence of the natural substrate or substrate-analogs.Our results suggest that local changes in the catalyticsite indeed affect the global conformation and stability ofthe dimeric protein. These results provide insights fornew alternatives to inhibit histamine production efficientlyin vivo.

Keywords: histidine decarboxylase; histamine; a-fluoro-methylhistidine; L-histidine methyl ester; pyridoxal phos-phate-dependent enzymes.

Mammalian histidine decarboxylase (HDC), the enzymeresponsible for the biosynthesis of histamine, is a pyridoxal5¢-phosphate (PLP)-dependent enzyme that belongs to theevolutionary group II of L-amino acid decarboxylases [1–3].Histamine is involved in several physiological responses(immune responses, gastric acid secretion, neurotransmis-sion, cell proliferation, etc.) and is also implicated in widelyspread human pathologies (inflammation-related diseases,neurological disorders, cancer and invasion) [4–8]. In spiteof the importance of these pathologies, HDC has not beenfully characterized, and important questions about theregulation of the enzyme expression, sorting, processing,structural characterization and turnover remain unan-swered [9–15].

Mature HDC purified from mammalian tissues has beenreported to be a dimer. Although the exact sequence of each

monomer is not known, it is generally believed that the74 kDa precursor is processed to a carboxy-truncated formof 53–58 kDa [16,17]. The N-terminus of the polypeptide(residues 1–480) exhibits a moderately high degree ofidentity with the porcine DOPA decarboxylase (DDC),another dimeric group II L-amino acid decarboxylase forwhich an X-ray structure has been solved [18]. Recently, wehave characterized the catalytic mechanism of a recombin-ant carboxy-truncated form of the rat enzyme (fragment1–512, also named HDC 1/512) [19], which shows kineticconstants similar to those of the mature enzyme purifiedfrom rodent tissues [16,17].

Mammalian HDC and DDC appear to share severalcatalytic features [19,20]. First, the PLP-enzyme internalSchiff base consists mainly of an enolimine tautomeric form(free holoenzyme). Second, Michaelis complex formationleads to a polarized ketoenamine form of the Schiff base.Third, after transaldimination, the coenzyme–substrateSchiff base exists mainly as an unprotonated aldimine.Finally, decarboxylation occurs and the free holoenzyme isrecovered after protonation and a reverse transaldiminationthat releases the amine product. In spite of these sharedfeatures, the following observations [19] suggest that keystructural differences must exist between the mammalianHDC catalytic site and those of the other group II L-aminoacid decarboxylases: (a) HDC is the least efficient enzyme ofits group; (b) mammalian HDC activity is less sensitive to thepresence of thiol reducing compounds in the medium thanother homologous and nonhomologous PLP-dependentdecarboxylases; and (c) transaldimination from the internalto the external aldimine involves a higher degree of rotationin the torsion angle (v) than those observed for otherhomologous enzymes. The last observation is in agreementwith the remarkably low catalytic efficiency of mammalian

Correspondence to F. Sanchez Jimenez, Departamento de Biologıa

Molecular y Bioquımica, Facultad de Ciencias, Universidad de

Malaga, 29071 Malaga, Spain.

Fax: + 34 95 2132000, Tel.: + 34 95 2131674,

E-mail: [email protected]

Abbreviations: DDC, aromatic L-amino acid decarboxylase or DOPA

decarboxylase (EC 4.1.1.28); DOPA, L-3,4-dihydroxyphenylalanine;

a-FMH, alpha-fluoromethylhistidine; a-FMHA, alpha-fluoromethyl-

histamine; HDC, histidine decarboxylase (EC 4.1.1.22); HisOMe,

L-histidine methyl ester; PLP, pyridoxal 5¢-phosphate.

Enzymes: aromatic L-amino acid decarboxylase or DOPA decarb-

oxylase (EC 4.1.1.28); histidine decarboxylase (EC 4.1.1.22).

*Present address: Advanced Science and Technology Laboratory,

AstraZeneca R & D Charnwood, Loughborough, UK.

(Received 7 August 2003, revised 9 September 2003,

accepted 12 September 2003)

Eur. J. Biochem. 270, 4376–4387 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03834.x

HDC, as this angle defines the degree of conjugationbetween the pyridine ring and the imine bond (maximum atv ¼ 0), which is important for catalytic efficiency of PLP-dependent enzymes [21]. In fact, the catalytic steps afterthe first transaldimination are rate-limiting in the entirecatalytic reaction of mammalian HDC [19].

On the other hand, the crystal structure of DDC hasshown that the catalytic sites of this enzyme are located inthe interface between monomers [18]. Assuming that thecatalytic sites of HDC are also in the interface betweenmonomers, we postulated the hypothesis that the observedhigh degree of rotation of the torsion angle v induced by thepresence of the substrate could produce a remarkableconformational change in the whole holoenzyme.

In this work, we have used biocomputational methodsto locate the catalytic center and predict the shape of thedimeric protein. The model reinforced our hypothesisproposed above. To further address this hypothesis, wehave characterized the protein conformational changesduring catalysis, by using a strategy similar to that usedpreviously for the catalytic mechanism characterization[19]. Substrate analogs capable of blocking the HDCcatalytic site at different catalytic steps are known, allowingthe detection and analysis of the respective conformationalstates of the protein during the reaction. Thus, electro-phoretic, chromatographic and spectroscopic analysescarried out with purified preparations of the recombinantHDC 1/512 version in the presence and absence of substrateor substrate analogs have allowed us to characterize theconformational changes of the holoenzyme whenever aPLP substrate- or PLP product-like adduct is inside thecatalytic center. We have also evaluated the stability ofthese conformational states against several agents thatdisrupt structure, i.e. detergent, thiol reductants, andtemperature.

Materials and methods

Biocomputational analyses

An initial model of the target protein (residues 5–479) wasgenerated from the rat HDC sequence (Swiss-Prot accessionnumber P16453) using the automated comparative proteinmodeling server SWISS-MODEL [22–24] in First Approachmode. The two pig DDC structures obtained from theProtein Data Bank (PDB ID 1JS3 and 1JS6) were used astemplates.

The docking program GRAMM [25] was used to build thestructure of the dimeric rat HDC from the coordinate fileprovided by SWISS-MODEL. A low resolution docking wasperformed with a grid step of 6.8 A and 20� rotationincrements. This type of docking is designed to overcomethe problems of conformational flexibility and induced fitmovements inherent to the formation of a protein–proteincomplex. The lowest energy docking solution was selectedas representative of the dimer structure. Secondary structureof the model was calculated with the DSSP program[26]. Energy minimization calculations were performed withthe program XPLOR [27] in an SGI Altix 3000 under GNU/LINUX REDHAT 7.2.

Three-dimensional visualization and analysis were per-formed with RASMOL [28] and SWISS-PDBVIEWER [22].

Molecular graphics were generated using MOLSCRIPT [29],RASTER3D [30] and PYMOL [31].

Recombinant HDC purification proceduresand enzyme activity assay

The DNA encoding for residues 1–512 of rat HDC [32] wassubcloned in the pET-11a vector (Novagen, USA). Therecombinant plasmid transformed into the Escherichia coliBL21(DE3)pLysS strain. Transformed cultures wereinduced to express the HDC 1/512, which was purified byapplying three chromatographic steps (Phenyl-SepharoseCL-4B, DEAE interchange, and hydroxyapatite). The finalpreparations were dissolved in 50 mM potassium phosphate,0.1 mM PLP, pH 7.0. Purity of the HDC 1/512 constructwas checked by Coomassie blue staining and Westernblotting, and was higher than 95% in the final preparations.HDC activity was assayed by following 14CO2 release fromL-His-[U-14C] (American Radiolabeled Chemicals, USA).Analogs and histidine were provided by Sigma-Aldrich(Spain). All of these procedures (overexpression, purifica-tion and analysis) are described extensively elsewhere [19].When required, the enzyme was concentrated in differentAmicon (USA) ultrafiltration systems (cut-off between 10and 30 kDa) depending on the initial volume. To avoidinterference by free PLP, the final preparation was subjectedto size-exclusion gel chromatography by using a SephadexG25 column or, alternatively, a Sephacryl HiPrep S-200column (Pharmacia Biotech, Sweden) in 50 mM potassiumphosphate buffer (pH 7) immediately before startingspectroscopic analyses.

Chromatographic analysis

Purified HDC preparations were incubated at room tem-perature in either the presence or absence of 1 mM alpha-fluoromethylhistidine (a-FMH) for 1 h. After incubation,protein was subjected to size-exclusion gel chromatographyon a HiPrep Sephacryl S-200 high-resolution column, pre-equilibrated with 50 mM potassium phosphate and coupledto a FPLC system (Pharmacia). Absorbance at 280 nm wasmonitored continuously to detect protein peaks. Mrs werecalculated after calibration of the column with the followingmolecular-mass standards (all from Sigma): alcohol dehy-drogenase (Mr 14 2000), bovine serum albumin (Mr

65 000), chymotrypsinogen A (Mr 25 000) and cyto-chrome c (Mr 12 400).

Electrophoretic and Western blotting analysis

Aliquots of the purified enzyme (1–3 lg) were incubated atroom temperature for 1 h in the presence of either 1 mM

histidine-analogs or 10 mM histidine, or in their absence(untreated enzyme). All solutions were adjusted to pH 7.Ten millimolar histidine (more than 20-fold the previouslyreported Km value) was chosen to maximize the percentageof enzyme taking part in the enzyme–substrate complex.When indicated, 2-mercaptoethanol was added after 55 minof incubation to yield a final concentration of 80 mM,followed immediately by loading buffers. For conventionaldenaturing SDS/PAGE experiments, the protocol describedby Laemmli was followed [33]. Samples were boiled and

� FEBS 2003 Conformational changes of histidine decarboxylase (Eur. J. Biochem. 270) 4377

stored at )20 �C until their use. However, for semidena-turing SDS/PAGE experiments, samples were not boiledand the loading buffers lacked 2-mercaptoethanol. Twodifferent loading buffers were used. Loading buffer A(pH 7.4) lacked SDS, while loading buffer B (pH 6.8)contained 0.7% SDS. Immediately after addition of therespective loading buffer, electrophoresis was performed at4 �C in a 7% acrylamide SDS-free PAGE gel (semidena-turing condition A) or in a 7% acrylamide 0.1% SDS/PAGE gel (semidenaturing conditions B), both lackingstacking gel. The running buffer always contained 0.1%SDS. To avoid problems with any minor contaminant bandin the purified preparations, electrophoretic results werevisualized by Western blotting, following a protocoldescribed elsewhere [15]. The anti-HDC K9503 serum wasgenerously supplied by L. Persson (Lund University,Sweden). Recombinant Strep-tag unstained standards161–0362 (Bio-Rad, USA) were used as references forelectrophoretic migrations. In each experiment, treatmentswere carried out in parallel on aliquots of the same purifiedenzyme preparations. The results of semidenaturing gelsshown here are representative of at least four differentindependent experiments.

Spectroscopic analysis

Absorption spectra were obtained using a HP8452A diodearray spectrophotometer (Hewlett-Packard, USA). Theacquisition time for each absorption spectrum was 2 s.Fluorescence spectra were obtained in a QuantaMasterSE spectrofluorimeter (Photon Technology InternationalInc., USA). Integration time was 0.1 sÆnm)1; three spectrawere averaged. CD spectroscopy was carried out with aJasco J-715 spectropolarimeter at a scan speed of50 nmÆmin)1; 10 spectra were averaged. Analogs wereused at the specified final concentrations. Unless otherwiseindicated, all spectroscopic measurements were carried outat room temperature. Fluorescence (300–400 nm, excita-tion at 275 nm) was not detectable from solutions of10 mM histidine or 1 mM analogs in an enzyme-freebuffer. CD signals (195–250 nm) were not detectable fromsolutions of 1 mM a-FMH, 14 lM PLP or both togetherin an enzyme-free buffer.

Results and discussion

Protein modeling of rat HDC dimer predictsthe location of its catalytic site

We have previously observed that transaldimination fromthe internal to the external aldimine of HDC involves ahigher degree of rotation in the torsion angle (v) than inother homologous enzymes [19]. This observation led usto postulate that the interaction of HDC with its substratecould induce significant conformational changes, at leastin the catalytic center environment. In addition, thisexperimental observation also indicated that some differ-ences must exist in the relative position of the cofactor–substrate adduct when compared with homologousenzymes.

The moderately high degree of identity between mam-malian HDC and DDC (> 50%), the published structural

models [2,3] and the reported crystal structure of porcineDDC [18], in combination make it possible to applycomparative modeling methods to overcome the lack ofreported crystal structures for mammalian HDC. Figure 1shows the lowest energy predicted 3D structure of the ratHDC monomer and its quaternary structure, obtained afterdocking and energy minimization calculations carried outas described in the Materials and methods section. Onethousand dimer structures were generated without anymanual fitting at all. At least the first 200 were examinedand found to be very similar. They are conformed astwofold axial symmetry dimers similar to those describedfor the crystal structures of pig DDC [18]. Nevertheless, itpredicts a more occluded catalytic center when comparedwith the DDC crystal structure. This is not surprising, asHDC has a more restrictive catalytic center, and it could besuspected from experimental data previously shown [19].

Figure 2 shows the alignment of rat HDC and pig DDCprimary sequences, as well as the distribution of a-helicesand b-sheets in both the crystal structure of pig DDC andthat estimated from the rat HDC 3D model. This figurestresses that the pattern of secondary structures in bothenzymes is very similar, in spite of their differences inprimary structure, as expected. The complete distribution ofconsensus secondary structure estimated from the model isas follows: 39% of a-helices, 9% of b-sheets, 12% of turns,21% of random coil and 19% of other structures. Theseestimations are similar to those obtained from rat HDCprimary sequence, and they are consistent with estimationsfrom far-UV CD spectra (controls at 20 �C in Fig. 9, andnot shown here to avoid redundancy).

In spite of the lack of overall sequence identity, acommon PLP-binding motif consisting of clusters ofconserved residues is present in decarboxylases belongingto groups I, II and III [2]. The PLP-binding site ofMorganella morganii AM-15 HDC was experimentallylocated in its K233 residue [34]. This lysine residue isextremely conserved, and corresponds to K303 of pig andrat DDC, also previously shown to play this role [18,35],and to K308 in rat HDC. A histidine residue, corres-ponding to H197 in rat HDC, also is strictly conserved ingroup II of mammalian L-amino acid decarboxylases, inwhich it seems to be stacked in front of the cofactorpyridine ring [18]. Thus, our model combined with thepreviously reported structural and mechanistic character-ization of these enzymes [19,20,36] allowed us to locatethe HDC catalytic center at the interface between themonomers (Fig. 1), as is the case of DDC [18]. One of themonomers (monomer A) would contain the major part ofthe catalytic site pocket, including K308 and H197 in ratHDC.

Figure 3 shows the most important residues close toH197 and K308, as predicted by our 3D model. Thepredicted catalytic center contains a number of polarresidues (D276, N305, H197 and K308). All of theseresidues are strictly conserved in mammalian DDC (seeFig. 2), where they take similar positions to thosepredicted in HDC. In mammalian DDC, D271 andN300 (counterparts of D276 and N305 in rat HDC) arepredicted to interact with the imidazole ring and thephosphate group of PLP, respectively [18,36,37]. It isnoteworthy that in spite of the high flexibility of the

4378 C. Rodrıguez-Caso et al. (Eur. J. Biochem. 270) � FEBS 2003

fragments (see Fig. 2) containing most of these residues,our prediction located them very close in space and atsimilar positions to those described for DDC [18].Therefore, it seems to be likely that they play a similarrole in mammalian HDC, delimiting what could betermed as the PLP interaction region (PLP-IR, seeFig. 3).

After formation of the holoenzyme, the substrate (histi-dine) should enter the catalytic site from the bottom part ofFig. 3 through a space delimited by the PLP-IR and aregion in which our model predicts the location of severalresidues of both monomers able to establish hydrophobicinteractions; for instance, Y84 (Fig. 3) and the fragmentPAL 85–87 from monomer A (the latter not shown in Fig. 3for clarity), and F331, I364 and L356 from monomer B.These predictions are in agreement with previous bio-physical and kinetic studies from our laboratory indicatingthat, in the internal aldimine form, the catalytic site of HDCis enriched in hydrophobic residues, leading to an enoliminetautomeric form of PLP [19]. A hydrophobic channel forthe substrate has also been proposed for DDC [38,39].

However, the specific hydrophobic residues of monomer Bcontributing to this region of DDC (I101 and F103, asdeduced from data in reference [18]) are different, asexpected from the structural differences between theirrespective substrates. It is also noteworthy that some ofthe closest hydrophobic residues of monomer B (forinstance, F331) are part of or close to the �flexible loop�described for mammalian DDC, which could not be solvedfrom the crystal structure (residues 328–339 in Fig. 2). In pigDDC, a conformational change of this loop in response tosubstrate binding has been demonstrated [18,37]. A similarrole of its counterpart in mammalian HDC in relation to theconformational change described in the present work couldbe suspected.

Finally, from this model we have predicted that theoccupation of the catalytic center by the polar substrate oran analog through a hydrophobic channel could inducedrastic conformational changes of the holoenzyme thatwould probably affect, at least locally, interactions at themonomer interface and vice versa. This reinforces ourstarting hypothesis.

Fig. 1. Three-dimensional model of rat HDC

structure. The model was generated from res-

idues 2–475 of the primary sequence, as des-

cribed in Material and methods section. (A)

and (B) Surface representations of the dimeric

form, one monomer in white and the other in

red. (C) Surface representation of one mono-

mer in white. The predicted interface between

monomers is shown in red. The active site

residues, K308 and H197, are shown in blue.

W and Y residues are depicted in green. In (A)

and (C), the white monomer is shown in the

same position. (B) is left-twisted around the

z-axis with respect to (A) to show the

localization of K308 and H197 within the

monomer interface. A double-headed arrow

in (A) indicates the maximum distance

determining the Stokes’ radius predicted for

the dimer.


Active HDC1/512 is a dimer and the presenceof a substrate analog in its active site diminishesits Stokes’ radius

HDC1/512 is a recombinant carboxy-truncated form of therat enzyme that we have previously used to study structure/function relationships of the mature HDC [10,13,14,19,40].We have recently shown that HDC1/512 has kineticconstants similar to those of the mature enzyme purifiedfrom rodent tissues [19]. Figure 4 shows the results of size-exclusion gel chromatography of purified HDC1/512. Amajor peak (Mr 107 000) was observed for the untreatedenzyme. Some inactive higher molecular weight HDCaggregates were detected by Western blots (data not shown).In fact, the purified enzyme preparations slowly tend toform inactive aggregates when incubated at room tempera-ture or higher (C. Rodrıguez-Caso, D. Rodrıguez-Agudo,

A. A. Moya-Garcıa, M. A. Medina, V. Subramanian &F. Sanchez-Jimenez, unpublished observations). Enzymaticactivity was only detectable in fractions corresponding tothe major peak. These results indicated that the quaternary

Fig. 2. Alignment of rat HDC and pig DDC sequences. The 5–479

fragment of rat HDC (Swiss-Prot accession number P16453) and pig-

DDC sequence from the Protein Data Bank (PDB ID 1JS6) were

aligned using the ProdModII method in Swiss-Model server (http://

www.expasy.org/swissmod/SWISS-MODEL.html). The secondary

structure of the crystallized pig DDC and that predicted from the rat

HDC 3D model are shown below the aligned sequences: h, helix;

s, sheet; ?, unpredicted conformation in crystallized pig-DDC.

Fig. 3. Structural neighborhood of the PLP-binding site. The most

relevant residues closer than 7 A to H197 or K308, as predicted by our

3D apoenzyme model, are depicted. The line delimits the PLP inter-

action region (PLP-IR). The putative entrance for the substrate

between the PLP-IR and the hydrophobic region is marked with a star.

Fig. 4. Size-exclusion gel chromatography of the free-holoenzyme and

the a-FMH-treated enzyme. Purified enzyme was incubated for 1 h in

the presence or absence of 1 mM a-FMH and submitted to size-

exclusion gel chromatography, using a FPLC system as described in

the Material and methods. No monomer was observed. Fractions 1 to

38 represented void volume. For the free-holoenzyme extracts, enzyme

activity was coincident with the major peak. Arrows indicate the peaks

of theMr standards: 1, alcohol dehydrogenase (Mr 142 000); 2, bovine

serum albumin (Mr 65 000); 3, chymotrypsinogen A (Mr 25 000); 4,

cytochrome c (Mr 12 400).


structure of the active recombinant purified enzyme used inthis work is, indeed, a dimer, as also deduced for the nativeenzyme purified from natural sources [17].

Figure 5 shows a scheme of the HDC reaction and thespecific steps interfered by the substrate analogs histidinemethyl ester (HisOMe) and a-FMH, deduced from previousreports in the literature [19,41]. HisOMe, a reversiblecompetitive inhibitor, blocks the reaction after formationof an external aldimine tautomeric form very similar to thatof the PLP–histidine adduct (Fig. 3 and [19]). By using thesubstrate analog a-FMH, the reaction can proceed (inclu-ding the decarboxylation step) to form a-fluoromethylhis-tamine (a-FMHA; Fig. 5 and [41]). In the case of fetal ratHDC, this reaction has been reported to proceed muchslower than with the natural substrate histidine [42].Nevertheless, after decarboxylation and elimination of thefluoride, a reverse transaldimination can occur, so that anenamine form of the product can either leave the catalyticsite or react again with the internal aldimine to form a PLP-adduct covalently attached to the catalytic center. It hasbeen proposed that the occurrence frequency of these twopossibilities depends on how long the enamine remains inthe active site and its rate of attack on the aldimine bond,and can be modified by slight differences in the position ofthe residues [41]. As covalent binding was proposed tooccur, at least partially, between PLP–a-FMHA derivativesand the enzyme, this analog is considered as a suicideinhibitor of PLP-dependent HDC [43]. Complexes III andVII in Fig. 5 would correspond to the major final formsstabilized at short-term in the catalytic site after thereactions with HisOMe and a-FMH, respectively.

Based on the shape of the predicted dimeric HDC(Fig. 1), a conformational change affecting the monomerinterface would change the Stokes’ radius of the protein, asthe major diameter of the dimer is predicted to be given by

the distance between the carboxy termini of bothmonomers (from the lower left to the upper right ofFig. 1B), which in turn is dependent on the dimerizationsurface conformation. Gel filtration is a validated methodto distinguish changes in the Stokes’ radius of an oligomericenzyme. Among the different compounds tested (thenatural substrate and substrate-analogs), a-FMH is theonly one that can accumulate a stable PLP-adduct cova-lently bound to the enzyme (Fig. 5), thus being able towithstand a gel filtration procedure. An apparent reductionof the Mr was indeed observed in the a-FMH-treatedsamples (Fig. 4), suggesting a treatment-induced change ofthe dimeric structure to a conformation with diminishedStokes’ radius.

Analog-treated HDC shows altered electrophoreticmobility under semidenaturing conditions

It is well known that quaternary structure of proteins isfrequently established, at least partially, through hydropho-bic interactions that can be weakened by SDS and otherdetergents. Thus, electrophoresis of the samples carried outin the presence of SDS as the only denaturing agent couldreveal: (a) reinforcements of monomer associations, as onlythe strongest associations could survive the denaturingagent; and (b) any change in the volume of a singlepolypeptide (or a polypeptide association). We analyzedHisOMe- and a-FMH-treated samples under the semide-naturing conditions described in the Material and methodssection. Figure 6 shows that treatments with analogs changethe relative electrophoretic mobility of untreated HDCunder semidenaturing conditions, supporting our hypothe-sis on global conformational changes of HDC induced bythe presence of analogs in the active center. Furthermore,these findings also seem to indicate that the analogs can

Fig. 5. Scheme of the HDC reactions with the

natural substrate histidine and the histidine-

analogs HisOMe and a-FMH. This scheme

was built from the major forms for each step

mentioned in the text deduced from the pre-

vious information ([18,19] and the present

results). The absorption spectrum maxima

described for the tautomeric forms mentioned

in the text are indicated in brackets. The pro-

posed major forms reached with HisOMe and

a-FMH are shown inside dashed boxes.

T, transaldimination steps.


induce changes of the enzyme to conformational states moreresistant to denaturation by detergents, especially in the caseof conformations adopted during the external aldimine state(HisOMe-treated samples).

Absorption spectra of HDC during reaction with histidineand analogs reveal details of the catalytic mechanism

Taking into account the extremely low reaction ratereported for mammalian HDC [19], and especially in thepresence of a-FMH [42], it is expected that different stepsof the reaction can be distinguished as a function of timein a conventional UV-visible spectrophotometer. Before

Fig. 6. Western blots of the free-holoenzyme and analog-treated samples

under different semidenaturing and denaturing conditions. Aliquots of

the same purified preparation were incubated for 1 h in the absence

(control, C) or presence of 1 mM histidine analogs (histidine methyl

ester, HisOMe; a-fluoromethylhistidine, a-FMH) and submitted to the

semidenaturing conditions A (A, loading buffer A) and B (B) and (C)

described in the Materials and methods section. In (C), samples had

been treated for 5 min with 2-mercaptoethanol. (D) corresponds to a

conventional denaturing SDS/PAGE electrophoresis. In this case,

molecular mass standards are shown as Mr · 10)3. In (A–C), bands

are designed according to their relative electrophoretic mobilities as F

(the fastest mobility), S (the slowest mobility) and I (intermediate

mobilities).

Fig. 7. Changes with time of the absorption spectra of HDC in the

presence of the natural substrate histidine or histidine-analogs. Con-

centrated, neutralized stocks (6.36 lL) of the natural substrate histi-

dine (A), HisOMe (B) or a-FMH (C) were added to 70 lL of a

13–14 lM solution of purified and gel filtered protein to reach final

concentrations of 10 mM histidine or 1 mM analogs.


addition of any substrate, we observed the same PLPabsorption profile previously reported for the free holo-enzyme (Fig. 7, untreated samples in all panels, and [19]):a major enolimine form (maximum at � 335 nm, complexI in Fig. 5) and a minor ketoenamine form (maximum at� 420 nm) of the internal aldimine. However, a fewseconds after substrate or analog addition, a new peakarose at 390 nm (Fig. 7, all panels), which must corres-pond to accumulation of enzyme molecules at the externalaldimine stage, as reported previously ([19], see alsocomplex III in Fig. 5). The peak was observed not onlywith histidine but also with both analogs, correspondingto their reported action mechanisms. After 1 min, this390-nm peak could still be observed in all cases. Asmentioned before, this external aldimine complex (com-plex III) is the final product of the reaction with HisOMe.In fact, after 5 min, the spectra of the HisOMe-treatedsamples had stabilized with the 390-nm peak as the majorand final one. In the reaction in the presence of an excessof the natural substrate histidine, a shoulder around430 nm is also observed (Fig. 7A), which must correspondto Michaelis complexes (complex II in Fig. 5) and/or toketoenamine forms of external aldimines (that is to say, to

other stages of the reaction), many of them having typicalabsorption maxima around 430 nm [44].

On the other hand, when using a-FMH, accumulationof a PLP-derivative with an absorption maximum at� 345 nm can be clearly observed after the reaction hadpassed through a maximum concentration of the externalaldimine complex (Fig. 7C). From the first minutes on,this peak became the major one clearly observed in thea-FMH-treated enzyme, suggesting that it corresponds toa major molecular form of the PLP–a-FMHA derivativethat was rather stable for at least the first hour oftreatment (complex VII in Fig. 5). Nevertheless, as theabsorption at wavelengths higher than 400 nm was evenincreasing with the treatment period, other noncovalentlybound PLP adducts cannot be ruled out. Bhattacharjeeand Snell [41], working with the bacterial M. morganiiHDC enzyme, proposed that the covalently bound adductcan be converted slowly into other PLP adducts withabsorption maxima higher than 400 nm (not shown inFig. 5), which can be removed from the catalytic center bydialysis and boiling. The absorption maximum observedat 345 nm, as well as the shape of the final spectra, areextremely similar to that reported [41] for the product of

Fig. 8. Fluorescence emission (excitation at 274 nm) of the free-HDC holoenzyme and the enzyme treated with the natural substrate or analogs. Ten

microliters of 50 mM potassium phosphate pH 7 (control condition), or 10 lL of concentrated neutralized stocks of the natural substrate histidine

(A), HisOMe (B) or a-FMH (C and D) were added to 90 lL of a 6.5–7 lM solution of purified and gel filtered protein to reach final concentrations

of 10 mM histidine or 1 mM analogs. Stability of the control spectra were assessed by three different determinations.


the enamine reaction with the internal aldimine, that is, aPLP-a-FMHA derivative covalently bound to the enzymeof Gram negative microorganisms (also Fig. 5, complexVII). As far as we know, this is the first time that thespectra of the PLP-adducts during the reaction of amammalian enzyme with a-FMH are recorded. Previousstudies of the reaction were carried out on partiallypurified extracts of the rat enzyme, working withradiolabeled a-FMH [42]. These authors deduced thatone-third of the decarboxylated a-FMH productsseemed to be covalently attached to the enzyme. Ourdata are also consistent with this proposal. As theinhibitor is in excess with respect to the enzyme, successivedecarboxylations would accumulate the covalent adduct inthe assay period, thus becoming the major form detectedin the spectra.

Summarizing, data shown in Fig. 7 reinforce our previ-ous findings on the tautomeric forms of the internalaldimine in the free holoenzyme [19] and the reactioncarried out with the suicide analog a-FMH [40].

The conformational changes induced in HDCby histidine and histidine-analogs involve alterationsin the environment of the aromatic amino acidresidues of the protein

Conformational changes of proteins can modify the intrin-sic fluorescence from their aromatic residues. Both HisOMeand a-FMH block the HDC catalytic center after the firsttransaldimination step (after external aldimine formation),leading to catalytic sites occupied by different PLP-adducts.HDC 1/512 contains 11 W and 13 Y residues. From them,six W and seven Y residues are predicted to be in themonomer interface (Fig. 1). These represent most of the W(86%) and Y (64%) residues predicted to be exposed to themonomer surface (seven W and 11 Y). Thus, it seemed likelythat a conformational change in the monomer interactionsurface could be reflected in the fluorescence measurementsof the free holoenzyme and the enzyme after addition ofhistidine and histidine analogs. An increase in W fluores-cence intensity is most commonly associated with reducedexposure of the W residues to the solvent, i.e. a transitionfrom a predominantly solvent-exposed to a more hydro-phobic situation brought about by a conformationalchange. Although an increase in interaromatic energytransfer is also possible, this is not the most likely reasonfor a fluorescence increase. Indeed, when there are severalaromatic residues in close proximity, interactions quenchingthe fluorescence tend to occur [45].

Figure 8 shows the fluorescence emission spectra (from300 to 400 nm, excitation at 274 nm) of HDC holoenzymeobtained before and after substrate or analog addition. In allcases, increases of fluorescence were observed to occur withinthefirstminuteafter thecompoundaddition, suggesting that,indeed, all of them are able to induce structural changesthat shield aromatic residues from solvent interactions. It is

Fig. 10. Thermal denaturation profile at 222 nm of the free HDC

holoenzyme and the analog-treated samples.Aliquots of a 3-lM solution

of purified and gel filtered enzyme were treated with or without the

histidine-analogs at 1 mM final concentration. CD spectra were

recorded after stabilization of the samples at the different assayed

temperatures. Stabilization times were 2–5 min.

Fig. 9. Thermal denaturation of the free HDC holoenzyme and the

a-FMH-treated enzyme.Aliquots of a 3-lM solution of purified and gel

filtered enzyme solution were treated (B) or not (A, free holoenzyme)

with 1 mM a-FMH. CD spectra were recorded after stabilization of the

samples at the different assayed temperatures. Stabilization times were

2–5 min.


noteworthy that these conformational changes take placewithin the time period in which most of the enzyme is passingthrough the external aldimine state, irrespective of thesubstrate or analog added (Fig. 7). At least with bothhistidine and HisOMe, a shift to the blue in the emissionmaximum could be clearly observed, supporting a transitionto a more hydrophobic environment.

On the other hand, an increased, red-shifted fluorescenceis observed in the a-FMH-treated enzyme after 30 min ofthe reaction (Fig. 8), when most of the enzyme is formingthe covalently bound adduct with the PLP-a-FMHAderivative (Fig. 7). These observations suggest that theconformational change involves alterations in the environ-ment of the aromatic amino acid residues of the protein.

Resistance of the different conformational statesto thermal denaturation

Results obtained with semidenaturing electrophoresis seemto indicate that histidine analogs can induce changes in theenzyme to conformational states more resistant to dena-turation by detergents. To test whether these analog-induced conformational states are also more resistant tothermal denaturation, we carried out CD analysis of HDCunder several treatments and at different temperatures.Changes in the secondary structure of the enzyme duringtemperature-induced unfolding can be deduced by usingthis approach. Figure 9 shows far-UV CD spectra(190–250 nm) of the free-holoenzyme (Fig. 9A) and thea-FMH-treated enzyme (Fig. 9B) incubated at differenttemperatures after treatment. Unfolding of the protein canbe deduced from changes in the spectra observed withincreasing temperatures (Fig. 9), as well as from the changesobserved in the ellipticity at 222 nm (Fig. 10). Nevertheless,these temperature-induced changes were more evident forthe free holoenzyme than for the a-FMH-treated enzymesample, indicating that the enzyme that had a covalentlybound PLP-adduct was more resistant to temperature-induced denaturation. Scarce 2D structural information canbe obtained from similar experiments made with HisOMe,due to the basal CD absorption of this compound.However, the observed increasing trend in the ellipticity at222 nm of the HisOMe-treated enzyme preparations(Fig. 10) suggests that the reversible inhibitor was not ableto protect the enzyme against thermal denaturation.

The increased resistance of the a-FMH-treated protein tothermal denaturation would indicate that the covalentbinding of the adduct to the catalytic center could fix somesecondary structure in the enzyme, suggesting severalinteraction points for the adduct within the catalytic centerin addition to those established by the internal aldiminealone. From the shape of the a-FMH-treated proteinspectra, stabilization of some a-helix by the cofactor adductcould be suspected (Fig. 9). It is noteworthy that most of thecatalytic site is predicted to adopt a-helix and random coilsecondary structures (predictions not shown, also derivedfrom Figs 1 and 2).

Concluding remarks

Since the initial suggestions by Pauling in 1948 and the laterformulation of the induced-fit hypothesis by Koshland, it is

well established that the interactions between an enzymeand its substrate induce conformational changes at theactive site. In most cases, these conformational changes areonly local and relatively small. However, for some enzymesthese changes may be remarkable. This seems to be the caseof mammalian HDC.

We had previously observed that transaldimination fromthe internal to the external aldimine of HDC involves ahigher degree of rotation in the torsion angle (v) thanthose observed in other homologous enzymes [19]. Thisobservation allowed us to postulate the hypothesis that theinteraction of HDC with its substrate could induce signi-ficant conformational changes, at least, in the catalyticcenter environment. By using biocomputational tools, wehave located the catalytic center of this enzyme in theinterface between the monomers (Fig. 1). Furthermore, wehave predicted that the occupation of the catalytic center bythe substrate or an analog could induce global conforma-tional changes in the intact holoenzyme, reinforcing ourstarting hypothesis. Evidence has also been obtainedsuggesting differences between these conformations beforeand after decarboxylation, revealed by using HisOMe anda-FMH, respectively.

As the catalytic site of HDC is located at the dimerinterface, small changes in the interaction surface betweenmonomers could affect the exposure of the catalytic pocketcontent to the medium. During reaction, PLP-substrateand PLP-product adducts are not covalently bound to theenzyme. Thus, it would make sense that conformationalchanges occur to keep the reaction intermediates within thecatalytic site, at least for several seconds, which is the timereported to complete the decarboxylation reaction of asingle histidine molecule [16,19,46].

It is worthwhile mentioning that some conformationalchanges have also been suggested for homologous enzymesduring the decarboxylation reaction. For instance, it hasbeen proposed that the fragment 328–339, which containsresidues proven to be important for the activity of theenzyme and which cannot be properly resolved by X-raydiffraction studies, could be a flexible part of the moleculethat changes its conformation during catalysis [18,37,38].More recently, Hayashi et al. [47] have reported animportant conformational change in aspartate aminotrans-ferase after substrate binding, which promotes the catalyticreaction, as it favors maximum imine–pyridine conjugation.Aspartate aminotransferase is also a dimeric PLP-depend-ent enzyme with a similar fold and some catalytic propertiesin common with both DDC and HDC [19,21,48]. The exactnature of the mammalian HDC conformational change isstill unknown; nevertheless, a process similar to thatoccurring in the transaminase could lead to a more severerotation in angle v up to negative values [19], so that theseconformational changes are related to the catalytic effi-ciency of the enzyme.

Our results indicate that mammalian HDC adopts, atleast two well-differentiated conformations during thecatalytic reaction. The one corresponds to the fully activeinternal aldimine of the enzyme, and the second takes placeduring the presence of a PLP-adduct (PLP-substrate orPLP-product) in the catalytic site. These conformationalchanges are part of the HDC reaction with its naturalsubstrate. The latter conformation represents inactive forms


of the enzyme, which occur during the rate-limiting steps ofmammalian HDC catalysis [19]. Therefore, we suggest thatthe full molecular characterization of these conformationalchanges could be useful to look for new strategies to inhibitspecifically and efficiently histamine production in vivo. Thisalternative strategy, blocking the enzyme in the second typeof conformation, would not necessarily have to involve theentrance of any inhibitor into the catalytic site, as theconformational change seems to affect the whole enzymestructure. In addition, the present results and experimentalapproaches could also be interesting for groups working inother enzymes with a similar folding model (i.e. L-aminoacid decarboxylases, amino transferases).

Acknowledgements

This work was supported by Grants SAF2002-2586 (MCYT, Spain),

REMA (ISCIII, Spain), Fundacion Ramon-Areces and Junta de

Andalucıa (CIV-267). CRC received a FPU fellowship from MCED

(Spain) and funds for a short stay in the Department of Molecular

Biology, Max Planck Institute for Biophysical Chemistry, Gottingen,

Germany. We are indebted to Dr T. M. Jovin (Max Planck Institute,

Gottingen) for accepting CRC in his department, to Dr J. L. Urdiales

(University of Malaga) and Dr J. V. Fleming (University of

Massachusetts Medical Center) for their valuable comments, and to

Drs J.A. Ranea and A. Valencia (National Centre of Biotechnology,

Madrid, Spain) for advice during 3D structure prediction of rat HDC.

Thanks are due to the Department of Architecture of Computers

(University of Malaga) for allowing us to get access to its computing

facilities.

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