THE Exploring the Pyridoxal 5 -Phosphate- CHEMICAL RECORD ... 200… · ABSTRACT: Pyridoxal...

275© 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

Exploring the Pyridoxal 5′-Phosphate-Dependent Enzymes

ANDREA MOZZARELLI, STEFANO BETTATIDepartment of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy

Received 9 August 2006; Revised 11 October 2006; Accepted 10 October 2006

ABSTRACT: Pyridoxal 5′-phosphate (PLP)-dependent enzymes represent about 4% of the enzymesclassified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reac-tions exploiting the electron sink effect of the pyridine ring, the conformational changes accompa-nying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral propertiesof the different coenzyme-substrate derivatives signaling the reaction progress, are some of the fea-tures that have attracted our interest to investigate the structure–dynamics–function relationships ofPLP-dependent enzymes. To this goal, an integrated approach combining biochemical, biophysical,computational, and molecular biology methods was used. The extensive work carried out on twoenzymes, tryptophan synthase and O-acetylserine sulfhydrylase, is presented and discussed as repre-sentative of other PLP-dependent enzymes we have investigated. Finally, perspectives of PLP-dependent enzymes functional genomics and drug targeting highlight the continuous novelty of an“old” class of enzymes. © 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 275–287; 2006: Published online in Wiley InterScience (www.interscience.wiley.com)DOI 10.1002/tcr.20094

Key words: cofactors; enzyme catalysis; immobilization; polarized spectroscopy; reactiveintermediates

The Chemical Record, Vol. 6, 275–287 (2006)

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� Correspondence to: Andrea Mozzarelli; e-mail: [email protected]

Introduction

Since the beginning of modern enzymology, pyridoxal 5′-phosphate (PLP)-dependent enzymes have attracted theinterest of many biochemists, rivaling with glycolytic and pro-teolytic enzymes, with mammalian aspartate aminotransferaseand bacteric tryptophan synthase (TS) among the most inves-tigated ones. This interest was supported by the large amountof protein available from organs like liver and heart, the exis-tence of mitochondrial and cytosolic isoforms, the availabilityof natural mutants, the complex chemistry of the catalyticmechanism, and the spectral changes of the coenzyme associ-ated to the enzyme catalysis.1 Today, PLP-dependent enzymes

are still a subject of many investigations, further triggered bythe outcome of functional genomics, the improvement ofcloning, expression and purification techniques, and the dis-covery of their involvement in several human diseases, thuscalling for specific inhibitors/drugs. They have been groupedinto three functional families on the basis of the carbon atomof the substrate involved in catalysis,2 and into five-fold typesbased on sequence and structure similarity.3 A key feature thathas emerged from the investigations of PLP-dependent

© 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

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enzymes is the versatility of the coenzyme in supporting verydistinct catalytic actions (Scheme 1).4–8 This, in turn, evidencesthe tight coupling between chemistry and protein structureand dynamics. Furthermore, PLP-dependent catalysis is dom-inated by a sequence of steps involving distinct intermediates,some of which are common to most of the members of thisenzyme family (Schemes 2 and 3). In the native enzyme PLP,bound via a Schiff base to an active site lysine, forms an inter-nal aldimine. In the presence of substrate or substrate ana-logues, catalysis proceeds via the formation of gem-diamine,external aldimine, quinonoid, ketimine, or aminoacrylateintermediates. Some of these species exist as an equilibriumbetween tautomeric forms, the enolimine, and the ketoe-namine, stabilized by a more apolar and polar microenviron-ment, respectively (Scheme 4). Distinct spectral properties areassociated to each catalytic intermediate, thus providing suit-

able signals to monitor the catalysis and to determine the cat-alytic mechanism (Table 1).1 In order to fully understand thecatalytic mechanism, which is based on the interplay betweenstructure, dynamics, and function of the coenzyme and theprotein matrix, a combination of approaches is required. Thesecomprise (i) X-ray crystallography to obtain the three-dimen-sional structure of the enzyme; (ii) molecular dynamics to trackatoms and protein segments during action; (iii) molecularbiology to generate site-specific mutants; and (iv) spectroscopictechniques to detect conformational changes and to determineequilibrium and kinetics parameters. During our studies, wehave applied, to some extent, all of these approaches. Further-more, we have also applied single-crystal polarized absorptionmicrospectrophotometry to tightly link the structural infor-mation obtained on a protein in the crystalline state to func-tion assayed in the same physical state, and to compare

� Stefano Bettati was born in 1966 in Parma, Italy. He received his M.S. degree from the Uni-versity of Parma in 1992, with a thesis on the allosteric regulation of the enzyme tryptohan syn-thase, and his Ph.D. degree in 1998 from the University of Modena, working on hemoglobinunder the supervision of Prof. Andrea Mozzarelli. He has been a postdoctoral fellow at the labo-ratory of Chemical Physics, National Institutes of Health, Bethesda, Maryland, under the direc-tion of Dr. William A. Eaton, from 1999 to 2000. Since 2001, he has been an assistant professorin Applied Physics at the Faculty of Medicine of the University of Parma, Italy. His current researchinterests are mainly in structure, function, and dynamics relationships of heme proteins and PLP-dependent enzymes, and protein immobilization techniques targeted to single molecule studies. �

� Andrea Mozzarelli was born in 1950 in Mantova, Italy. He received his degree in Chemistry in1974. He is Full Professor of Biochemistry at the Faculty of Pharmacy, University of Parma. Aswinner of a Fogarty fellowship, in 1984–1985 he carried out studies on hemoglobin S polymer-ization in the Laboratory of Chemical Physics, directed by Dr. William A. Eaton, at the NationalInstitutes of Health, Bethesda, Maryland. His main research interest is the elucidation of structure–dynamics–function relationships of hemoglobins, pyridoxal 5′-phosphate (PLP)-dependent enzymes and green fluorescent proteins. Protein immobilization via crystallization orencapsulation in silica gels has been exploited as a novel tool to select and isolate distinct tertiaryand quaternary conformations, analyzed by spectroscopic and microspectroscopic methods. Mod-eling and advanced computational methods are applied to determine free energy of ligand–proteininteractions and to predict the affinity of designed protein ligands. He is a member of the Interna-tional Advisory Board for the Vitamin B6 and Quinoproteins. He has been the organizer of theinternational courses “From Structural Genomics to Drug Discovery,” held at the University ofParma, Parma, Italy, in 2000, 2002, and 2004. He was the cochairman of the conference on“International Visions on Blood Substitutes” that was held in Parma, Italy, in September 2006. �

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functional properties in solution and in the crystalline state.9,10

We have recently also exploited the approach of protein encap-sulation in wet, nanoporous silica gels in order to isolatedefined conformational states and develop stable biosensorsand bioreactors.11,12

In this work, we will describe some of the PLP enzymesthat we have investigated over the years, pointing out the bio-logical questions we have addressed and the approach we haveused, with the goal to stimulate further research on the intrigu-ing world of PLP catalysis. Finally, we will provide some perspectives, from our personal point of view, on the mostadvanced investigations on PLP-dependent enzymes: thedigging out of the information generated by sequence analysisof genomes, the characterization of enzymes involved in dis-eases, and the comparison of folding mechanisms of PLPenzymes within the same fold type and among distinct foldtypes.

TS

Bacterial TS is an α2β2 complex that catalyzes the last two steps in the biosynthesis of L-tryptophan.13–16 Indole-3-glycerol phosphate is cleaved in the α-active site to generateglyceradehyde-3-phosphate and indole. The latter is subse-quently channeled via a hydrophobic tunnel to the β-active sitewhere it is combined with L-serine to form L-tryptophan. Thischanneling, which avoids the escape of indole to solution, rep-resents the first detected example of intramolecular channel-

ing.17 The structure of the wild-type TS and several mutantshas been determined.17–27

The reaction at the β-active site is carried out on the cofac-tor PLP, bound to a lysine via a Schiff base. The reaction proceeds through the formation of several intermediates, char-acterized by distinct absorption and emission properties(Scheme 2; Table 1). The α- and β-subunit activities are reci-procally regulated through the stabilization of a catalyticallyinactive “open” state or a catalytically active “closed” state ofboth the α- and β-subunits.28–30 Depending on the catalyticintermediate present in the active sites, regulatory signals aregenerated that affect the opposite subunit, thus finely tuningthe subunit catalytic activity and keeping them in phase.

Functional Properties of TS

The catalytic process carried out by TS from Escherichia coliand Salmonella typhimurium has been thoroughly investi-gated.13,15 We have concentrated our studies on the latterbecause its structure was the first to be determined. The cat-alytic mechanism, as derived from steady-state and presteady-state kinetic measurements, is reported in Scheme 2. We havefound that the equilibrium distribution between the externalaldimine and the α-aminoacrylate Schiff base is not onlyaffected by α-subunit ligands but also by pH, monovalent ions,and temperature,31,32 thus indicating that several “effectors” areable to modulate the equilibrium between open and closedsubunit conformations. Low pH, cesium ions, and α-subunitligands stabilize the α-aminoacrylate Schiff base (closed con-


Scheme 1. Catalytic versatility of pyridoxal 5′-phosphate (PLP)-dependent enzymes (adapted from Ref. 8).



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formation), whereas high pH, sodium, and potassium stabilizethe external aldimine (open conformation). In particular, wehave characterized by presteady-state single wavelength andrapid scanning stopped-flow measurements the pH depen-dence of the first phase of the β-replacement reaction, i.e., theβ-elimination step,33 and we are currently analyzing the secondphase, i.e., the β-addition step. We have found that the reac-

tion is controlled by groups with two apparent pKa values ofabout 6 and 9, in the absence and presence of sodium andpotassium ions, and a single pKa value of 9 in the presence ofcesium ions. By combining kinetic and structural data on thecatalytic intermediates, the pKas were tentatively assigned toAsp305β and Lys87β. A further steady-state and presteady-state kinetic investigation is in progress aimed at measuring the

Scheme 2. Catalytic mechanism of the β-reaction catalyzed by tryptophan synthase.


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isotopic effect at different pH values using deuterated L-serine.34

Protein Function in the Crystal

Determining the three-dimensional structure of the enzyme atdifferent stages of the catalytic process is an important com-ponent of the effort to understand the structural basis of theallosteric regulation of TS. Following the first report on thestructure of the internal aldimine,17 studies were carried outinvestigating the reactivity of TS in the crystal by polarizedabsorption microspectrophotometry, in order to determine theexperimental conditions for the selective accumulation of dis-

tinct catalytic intermediates, eventually suitable for the X-raycrystallographic analysis.19,32,35,36 Polarized absorption spectrawere recorded on crystals of TS suspended in a medium con-taining L-serine at different pH levels, in the absence and pres-ence of monovalent cations, and in the absence and presenceof the α-subunit ligand glycerol-3-phosphate. pH, monovalentcations, and α-subunit ligands were demonstrated to affect theequilibrium distribution of intermediates in the crystal as insolution. The last catalytic intermediate awaiting structuraldetermination is the quinonoid formed in the reaction of theα-aminoacrylate Schiff base with indole. This species is formed


Scheme 3. Catalytic mechanism of the reaction catalyzed by O-acetylserine sulhydrylase.

N

O

NH

COORH

NH H

O

NH

COORH

Enolimine Ketoenamine

Scheme 4. Ketoenamine and enolimine tautomers of the external aldimineSchiff base.

Table 1. Spectral properties of selected pyridoxal 5′-phosphateenzyme derivatives.

Absorbance FluorescenceSpecies (λmax, nm) (λmax, nm)

Protonated internal 330–340a, 412–425b Low intensityaldimine

External aldimine 330–340a, 420–430b 500c

Quinonoid 470–510 Not determinedα-Aminoacrylate 340–360a, 460–470b Low intensity

[a]Enolimine tautomer.[b]Ketoenamine tautomer.[c]λexc = 420–430nm.



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only transiently in solution and does not accumulate appre-ciably in the crystal. However, metastable analogues of thisquinonoid species were obtained by reacting the α-aminoacry-late Schiff base with nucleophiles such as β-mercaptoethanol,indoline, phenylhydrazine, and small organic amines.36,37

Attempts to determine the structure of the quinonoid speciesof TS have been carried out, but no structure is available yet,likely due to the intrinsic photolability of the quinonoidspecies, even under cryogenic conditions.

Dynamic Properties of the a2b2 Complex

In order to determine the enzyme flexibility at defined sitesand to detect ligand-induced conformational changes associ-ated to regulatory signals, steady-state and time-resolved fluo-rescence and phosphorescence emissions and 31P NMR weremeasured for the unique tryptophan residue of TS, Trp178βlocated at the subunit interface in the helix 6 of the β-subunit,and for the coenzyme, at different stages of the catalyticpathway, in the absence and presence of monovalent cationsand α-subunit ligands.38–41 It was found that the mobility ofTrp178β is significantly affected by α-subunit ligands as wellas by the catalytic intermediates stabilized at the β-active site,suggesting that this part of the protein is involved in inter-subunit regulatory signals.38,39 This hypothesis was further val-idated by both X-ray and functional studies.22,42 The changesof 31P NMR of the phosphate of the coenzyme, at differentstages of the catalytic pathway, were attributed either to achange in coenzyme flexibility or to the coenzyme protonationstate.41

Intersubunit Allosteric Communication

A key feature of TS is the presence of two distinct active sites,separated by about 20Å. In order to ensure that the overall cat-alytic process is productive and efficient, cross talks betweensites are operative. There are two distinct pathways of inter-subunit communication. The first one is predominantlyinvolved in the reciprocal subunit activation, i.e., the catalyticactivities of isolated subunits are 30- to 100-fold lower thanthose in the α2β2 complex. This communication involves theso-called COMM domain of the β-subunit that, via the β helix6, interacts with α loop 2, containing α-active site residues.22

Mutants at this interface alter the catalytic activity withoutaffecting the allosteric regulation.43 The second pathway is pre-dominantly dedicated to the active sites catalytic activity fine-tuning. In fact, ligands of one subunit control the open-closedconformation equilibrium of the other subunit. This commu-nication is achieved via the interaction between Gly181 of theα loop 6 and Ser178 of β helix 6 (Fig. 1). It has been proventhat the occupancy of the α-active site by α-subunit allostericligands triggers the stabilization of the closed state of the α-subunit through the formation of a single hydrogen bond

between the NH of αGly181 and the carbonyl oxygen ofβSer178.22,26 Several mutants of both residues were preparedand their functional and regulatory properties character-ized.44,45 Furthermore, limited proteolysis experiments on thewild-type enzyme and mutants were carried out under differ-ent experimental conditions,45 supporting the notion thatwhen the hydrogen bond between αX181 and βX178 isimpeded, α loop 6 is in the open conformation and regulatorysignals to the β-active site are knocked out. The structuralinvestigation of one of the mutants in the absence and pres-ence of α-subunit ligands26 further strengthens this conclusion,showing the conformation of the α loop 6 in the closed state.In the open state, the α loop 6 is undetectable by X-raymethods due to its mobility. To overcome this limitation, molecular dynamics simulations were carried out on the wildtype and mutants, in the absence and presence of α-subunitligands.46 The simulated conformations of the open state of αloop 6 correlate with the experimentally determined extent oflimited proteolysis and with the strength of the hydrogen bond between αX181 and βX178, evaluated by the softwareHINT (Tripos, Inc., St. Louis, MO),47–49 and allow to explainthe reduced activity of the α-active site for some of themutants.

Computer-Assisted Design of a-Subunit Ligands andAllosteric Effectors

Computational methods are used in biology for sequencealignment, molecular dynamics simulations, and homology

Fig. 1. Close-up view of the interaction between the α loop 6 and β helix 6of the tryptophan synthase α2β2 complex. Several hydrogen bonds (yellowdashed lines) are formed among residues of the α loop 6 and a single hydro-gen bond is formed between αGly181 and βSer178.


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modeling. We have applied computational procedures to thedesign of new inhibitors of the α-subunit.50 Such compoundswould feature practical applications being TS, an enzymepresent in bacteria and plants but not in vertebrates, a poten-tial target of pesticides. Our starting structure was that of thecomplex with the α-subunit ligand and allosteric effectorindole-3-propanol phosphate. By several structural modifica-tions, guided by the evaluation of the interaction energy andvolume occupancy, a series of indole-acetyl amino acids wasproposed as potential ligands. These ligands have in commonthe substitution of the phosphate moiety, present in the naturalsubstrate indole-3-glycerol phosphate and in the allostericeffectors indole-3-propanol phosphate and glycerol-3-phosphate, with a carboxylate group. The characterization of the binding affinity and regulatory properties indicated that indole-3-acetyl-glycine (IAG), indole-3-acetyl-aspartate(IAD), and indole-3-acetamide are both α-subunit ligands andallosteric effectors, whereas indole-3-acetyl-valine (IAV) andindole-3-acetyl-alanine are only α-subunit ligands. The mod-eling offers an explanation to this distinct behavior, with thelatter two compounds that do not favorably interact withαSer235, a residue that was proposed to be critical for allo-steric regulation. The three-dimensional structure of the α2β2

complex in the absence and presence of IAD, IAG, and IAVprovided convincing evidence of the different roles played bythese α-subunit ligands.26

O-acetylserine Sulfhydrylase (OASS)

OASS51 belongs to the fold type II and the β functional familyof the PLP-dependent enzymes, together with, among others,the β-subunit of TS, cystathionine β-synthase, and threoninedehydratase. OASS catalyzes the last reaction in the biosyn-thesis of cysteine in bacteria and plants, a β-replacement reac-tion that leads to the formation of cysteine from O-acetylserine(OAS) and sulfide, through a Bi Bi ping-pong kinetic mecha-nism (Scheme 3). The structure of the functional homodimerhas been solved in different conformations, in the absence andpresence of substrate analogues and allosteric effectors.52–54

Binding of L-cysteine, or other substrate analogues, such as L-serine and methionine, is followed by the formation of theexternal aldimine of PLP that is, in turn, associated with a con-formational change from an open (inactive) to a closed (active)structure. This structural transition is likely triggered by thebinding of the α-carboxylate of the amino acid substrate to anα-carboxyl subsite in the enzyme active site.54 Binding of chlo-ride to an allosteric anion-binding site, located at the dimerinterface, stabilizes a conformation that differs both from theopen conformation of the internal aldimine and the closed one of external aldimine.53 In such state, the formation of theexternal Schiff base and thus the subsequent chemistry are

inhibited. The authors suggest that the chloride ion maybehave as an analogue of sulfide, the physiological inhibitor ofOASS.

Protein Function in the Crystal

We have carried out a detailed characterization of the func-tional properties of OASS in the crystalline state by single-crystal polarized absorption microspectrophotometry.55 Theinvestigation was carried out on three different crystal forms,including the one used to determine the three-dimensionalstructure. Only one of the crystal forms appeared to beendowed with full catalytic competence (Fig. 2), despite sub-strate dissociation constants much higher than in solution,probably due to lattice forces limiting the structural flexibilityrequired by the open/closed transition. This result highlightsthe importance of carrying out functional studies in the crystalto assess the catalytic competence of the crystal structure andto validate protein structure–function relationships, most oftenbased on the comparison of functional data collected in solu-tion and structural information obtained in the crystallinestate. In the latter, lattice forces restraints or the selection ofspecific conformations, not necessarily those prevailing in solu-tion, might mislead about the identification of active siteresidues and structural changes that are relevant to function.The microspectrophotometric studies on OASS also allowedto define experimental conditions for the accumulation of cat-alytic intermediates in the crystal suitable for crystallographicanalyses, a necessary requisite for the elucidation of the cat-alytic pathway at the molecular level.


350 400 450 500 5500.0

0.5

1.0

1.5

Wavelength (nm)

Abs

orba

nce

E//a

E//b

Fig. 2. Polarized absorption spectra of O-acetylserine sulhydrylase (OASS)crystals in the presence of the natural substrate OAS. Spectra are recorded withthe electric vector of the linearly polarized light parallel to either the a or bcrystal axis. Crystals of the internal aldimine (solid lines) were suspended insolutions containing increasing concentrations of OAS: 0.33 (....), 1.32 (– .. –),5.5 (–––), and 18mM (– .– .–).



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Structural and Dynamic Information from Fluorescenceand Phosphorescence Measurements

Before the three-dimensional structure of OASS was solved byX-ray diffraction, several studies took advantage of the lumi-nescence properties of the coenzyme PLP and tryptophanresidues to gain information on protein flexibility and dynam-ics during catalysis. Fluorescence energy transfer to PLP, occur-ring mainly from one of the two tryptophans present in eachOASS-A monomer, is extremely sensitive to donor-acceptordistance, and provides an additional probe to investigate con-formational changes. Phosphorescence studies56 provided thefirst experimental evidence that the two tryptophans are dif-ferently exposed to the solvent and are quenched at a differentextent by energy transfer to PLP. The authors correctly pre-dicted the tryptophan-PLP distance, and the occurrence ofconformational changes in the tryptophans regions (i.e., about25Å away from the active site) upon binding of cofactor, sub-strates, and protons. We extended the investigation to steady-state and time-resolved fluorescence of tryptophans and PLP,both in the presence and absence of substrates and substrateanalogues.57–59 In the case of the internal aldimine of OASS,structured fluorescence emission and decays have been attrib-uted to an equilibrium between the enolimine and ketoe-namine tautomers of the coenzyme, and a dipolar speciesformed upon proton dissociation in the excited state.58 Thistautomeric equilibrium is altered by the addition of acetate,the first product of OASS reaction that actually binds as adead-end inhibitor at the α-carboxylate subsite. Acetatestrongly favors the ketoenamine tautomer, likely through thestabilization of a more polar active site microenvironment.

The reaction with the substrate OAS proceeds via thetransient formation of an external aldimine and a stable α-aminoacrylate intermediate (Scheme 3). Stable external aldi-mine species are obtained upon reaction with the productL-cysteine or the product analogue L-serine. Steady-state andtime-resolved fluorescence studies57 indicate that different cat-alytic intermediates stabilize different protein conformationsand a different equilibrium between tautomers. The externalaldimine is characterized by a compact structure, somewhatsimilar to that induced by the binding of acetate in the inter-nal aldimine.58 This closed conformation likely favors thecorrect alignment of catalytic residues and protects bound sub-strates from interactions with the solvent. The α-aminoacry-late intermediate is characterized by a more flexible active siteconformation, as indicated by time-resolved fluorescenceanisotropy, that might favor the release of the nucleophilicproduct and binding of the nucleophilic substrate in thesecond part of the reaction. Because of the energy transfer toPLP, in the presence of the substrate OAS or its analogue β-chloro-alanine, or the products or analogues acetate, L-cysteineand L-serine, also tryptophan fluorescence emission and

lifetime decays are significantly affected by variations of thetautomeric equilibrium.59 This equilibrium, in turn, reflectschanges in the active site polarity determined by conforma-tional changes like those collectively indicated as open/closedtransitions.

The molecular heterogeneity and tautomeric equilibriumof OASS internal aldimine have been investigated at a singlemolecule level by two-photon excited fluorescence fluctuationspectroscopy.60 The number and the fluorescence brightness of excited protein molecules were separately determined, yield-ing a relative abundance of 4 : 1 for the enolimine and ketoenamine tautomers, respectively, whereas the ratio of two-photon cross sections is reversed with respect to single-photonexcitation. We are currently extending the single-moleculestudies to fluorescence anisotropy measurements on dilutedprotein solutions and on OASS encapsulated in silica gels, bothfor the internal and external aldimine, to detect finer hetero-geneity effects possibly due to conformational substates.

Thermodynamic Stability, Folding Mechanism, and theRole of Conserved Residues

The common structural, dynamic, and functional motives ofPLP-dependent enzymes, characterized by a large variation inprimary sequence and substrate and reaction specificity, makethis superfamily of enzymes an interesting system to probe therelationships among sequence, coenzyme interactions, func-tion, and folding mechanism. We investigated the thermody-namic stability and unfolding mechanism of OASS usingseveral spectroscopic techniques: absorption, fluorescence, cir-cular dichroism, 31P NMR, time-resolved fluorescence and flu-orescence anisotropy, and photon correlation spectroscopy.61,62

The multiple spectroscopic probes allowed to monitor the con-formational state of several distinct regions of the proteinduring the unfolding process, showing that, different fromother PLP enzymes,61 OASS undergoes extensive disruption ofnative secondary and tertiary structure before monomeriza-tion. This is probably due to the very strong dimer interface.An interesting finding is that the coenzyme not only confers a higher thermodynamic stability to the holoenzyme withrespect to the apo- form, but, even in the absence of denatu-rant, it is necessary for the attainment of the native confor-mation. Circular dichroism spectra indicate, in fact, that holo- and apo-OASS are endowed with secondary structuresthat differ both quantitatively and qualitatively. The analysisof fluorescence lifetime decays of the two tryptophans ofOASS-A,62 which are located in different domains, indicatedthat despite the coenzyme being positioned at the interfacebetween the two domains, the different pattern of interactionsaccounts for a markedly stronger stabilizing effect on the C-terminal domain with respect to the N-terminal one. This dif-ferential stabilization is probably of significant functional


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relevance, considering the well-assessed compensation betweenthe thermodynamic stability and the structural plasticityrequired by enzyme function and regulation. Indeed, in thecase of holo-OASS, the formation of the external aldimineupon substrate binding implies an open/closed transitionwhere a subdomain belonging to the N-terminal domainundergoes the larger movement. The very modest overall sta-bility of OASS, of the order of less than 3kcalmol−1, appearsto be at least partially compensated by kinetic stability. Thevery low unfolding rates63 minimize the probability of theprotein to sample aggregation-prone unfolded or partiallyfolded states, preserving the required conformational flexibil-ity. We are currently extending our investigation on the kineticand thermodynamic stability of different catalytic intermedi-ates of OASS-A, on the B isozyme, endowed with a 60%sequence similarity with respect to OASS-A, and on the β-subunit of TS, belonging to the same fold type and functionalfamily.

Both tryptophan residues of OASS-A are largely exposedto the solvent, an uncommon location for large hydrophobicresidues in soluble proteins. Only one of them, Trp161, ishighly conserved among eukaryotes and prokaryotes OASSsequences. We characterized the effect of single tryptophan totyrosine mutations on structure, function, and stability.64 Themutations do not significantly alter the enzyme secondarystructure but affect the catalysis, with a predominant influenceon the second half reaction. The mutation of the poorly con-served Trp50 has been shown to dramatically alter the proteinstability and unfolding mechanism, likely through the desta-bilization of the subunit interface. The Trp161Tyr mutation,in the C-terminal domain, does not significantly change theunfolding mechanism and overall stability of OASS, indicat-ing that the evolutionary conservation of Trp161 is not asso-ciated to folding. However, acrylamide quenching dataindicate that the mutation produces a reduction in the acces-sibility of the active site. Together with the observed effects onthe reaction kinetic parameters and the local stability of the N-terminal domain, these results indicate that the Trp161Tyrmutation stabilizes a partially closed conformation of OASS-A. This suggests that the conserved Trp161, located on a flatsurface of the protein, might be involved in the pathway ofregulatory interprotein communications occurring upon theformation of the bienzyme complex between OASS and serineacetyltransferase (SAT). Experiments on the effect of the inter-action between SAT and the W161Y mutant of OASS A arecurrently under way.

Cell Biochemistry and Interactions With SAT

OASS plays a key role in sulfur metabolism, controlling thecysteine biosynthetic pathway both directly and indirectly. Adirect control is exerted through the formation of bienzyme

complexes with ATP sulfurylase and SAT, although it must benoted that a substantial fraction of OASS activity in the cell isnot found in association with SAT. The latter complex, group-ing the last two enzymes in cysteine biosynthesis, is usuallyreferred to as cysteine synthase. Moreover, the OASS substrate,OAS, and its product, L-cysteine, function as reporters ofsulfur supply to the cell and can modulate the activity and theexpression of the enzymes of the cysteine regulon, so that cys-teine biosynthesis is modulated at different levels: transcrip-tional control, activity regulation via feedback inhibition, andprotein–protein interactions. Very recently, our group con-tributed to the characterization of the effects of the formationof the cysteine synthase complex on the functional propertiesof OASS, which have been investigated exploiting the fluores-cence properties of PLP.65 Both SAT and its isolated C-terminal decapeptide bind to the α-carboxyl subsite of OASS,with dissociation constants in the low nanomolar and hun-dreds of nanomolar range, respectively. Binding triggers thetransition from an open to a closed conformation, indicatingthat SAT can inhibit the catalytic activity of OASS in twoways, by competing with the substrate OAS for binding to theenzyme active site and by stabilizing a closed conformationthat is less accessible to the natural substrate. The work byCampanini et al.65 also provided the first direct determinationof the stoichiometry of binding of SAT to OASS, clearly indi-cating that two dimers of OASS bind to one hexamer of SAT.

In bacteria, two isoforms of OASS, OASS-A, and OASS-B are known. Most structural and functional studies have beencarried out on OASS-A, while little is known on the biologi-cal role of OASS-B. We are currently investigating the func-tional properties, stability, and interactions with SAT of the Bisomer of OASS66 and working on the design of selectiveinhibitors of either OASS-A or OASS-B.67

Reactivity of PLP-Dependent Enzymes in theCrystalline State

Single-crystal microspectrophotometry has been used duringthe years to assess the catalytic competence of several PLP-dependent enzymes whose structures were determined by X-ray crystallography, and to define the experimental conditionsfor the accumulation of catalytic intermediates, eventually suitable for X-ray analysis.9,10 Among these enzymes are mito-chondrial aspartate aminotransferase,68,69 serine hydromethyl-transferase,70 cystathionine-β-synthase,71 cysteine β-lyase(C-DES),72 aminobutyrric acid (GABA) aminotransferase,73 L-amino acid decarboxylase,74 tyrosine phenol lyase (TPL) andtryptophan indole lyase (Trpase),75,76 TS,19,32,35,36 and OASS.55

The results of the investigations on the latter two enzymes havebeen already discussed (see previous discussion).

In the case of aminobutyrric acid aminotransferase, thepolarized absorption spectra of the enzyme crystals in the pres-




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ence of either γ-ethynylGABA or the antiepilepsy drug viga-batrin allowed to direct the interpretation of the mechanismof inhibition, correlating structural data and functional data insolution.73

For cystathionine β-synthase, a remarkable and unex-pected result was the possibility of reversibly removing theheme cofactor in the crystalline state,71 a process that in solution leads to enzyme aggregation and inactivation. In the des-heme enzyme, it was possible to characterize the PLP-dependent enzyme reactivity by monitoring the coen-zyme spectral properties, usually masked by the highly absorb-ing heme. Thus, the crystalline environment allows to carryout experiments, which in solution, are prevented. It wasfound that the enzyme in the crystal forms a stable α-aminoacrylate intermediate, as observed in the E. coli enzyme,that does not bind the heme, indicating the ability to catalyzethe β-elimination. The disappearance of the α-aminoacrylatespecies in the presence of homocysteine suggests that the crys-talline enzyme is also able to carry out the β-replacement.

PLP Enzymes Encapsulated in Wet NanoporousSilica Gels

The last decade experienced an impressive development in the application of sol-gel materials at the interface withbiology.12,77,78 In particular, novel synthetic approaches, withmild physical and chemical conditions, allowed the encapsu-lation in wet, nanoporous silica gels of proteins (enzymes, anti-bodies, and heme proteins), nucleic acids, polysaccharides,phospholipids, as well as viable whole cells. Protein-dopedsilica gels can be manufactured with different sizes and geom-etry, and the gel optical quality makes them suitable for thedetection of signals by spectroscopic techniques. The applica-tions span from bioactive materials and controlled drug releasesystems, to the design of bioreactors and biosensors, to bio-physical studies on encapsulated proteins. The nanoporousstructure makes the protein active sites accessible to smallreagents that diffuse in the solvent phase, so that the spectro-scopic, dynamic, and functional properties of the entrappedproteins, individually caged in the gel, are usually maintained,while all interprotein interactions, including aggregation, areprevented. A striking feature of protein encapsulation in silicagel, relevant for biochemical and biophysical investigations, isthe ability to select defined conformational states of proteinsby decreasing by orders of magnitude the rate of conforma-tional relaxations,79–85 or to bias under equilibrium conditionsa preexisting distribution of conformations.86,87 The effects ofencapsulation on the kinetics and thermodynamics of struc-tural relaxations are mediated by the peculiar property of thegel pores to reduce the accessible volume and to perturb thesolvent structure, effective viscosity, and activity of solvent and

solutes. Notably, these aspects reproduce in vitro many prop-erties of the crowded and confined environment experiencedby proteins in vivo, and, thus, can allow to unveil biologicallyrelevant structural and dynamic properties escaping detectionin the diluted solutions normally used for biophysical investigations.

Most of the previous works on encapsulated enzymesrevealed a significant reduction of the catalytic efficiency.12,88–90

However, most of these studies lack of a detailed characteriza-tion of the single catalytic steps, and the observed reduced cat-alytic rates could be limited by substrate diffusion within gelsof uncontrolled thickness. We have measured, in solution andin the gel, the catalytic activity and equilibrium distribution ofcatalytic intermediates of TS,86 TPL, and Trpase.87 The lattertwo enzymes catalyze the β-elimination reaction of L-tyrosineand L-tryptophan, respectively, to form pyruvate, ammonium,and either phenol or indole, and despite a high structural andfunctional similarity, are extremely specific for their respectivephysiological substrates. We have found that the three enzymesare catalytically competent in the gel, despite an altered distri-bution of catalytic intermediates and coenzyme tautomers,slow attaining of steady state, and a conspicuous reduction ofthe specific activity (Table 2). For TS, TPL, and Trpase, thetransition between open (inactive) and closed (active) states ofthese proteins accompanies substrate binding and the catalyticcycle, and all the observed results can be explained with theselective stabilization of tertiary conformations and/or adecrease of the rate of open-closed transitions. Our currentgoal is to modify gelification conditions in order to pursue anincreased and reproducible catalytic efficiency for biotechno-logical applications, a result brilliantly achieved with encapsu-lated lipases.91

Perspectives on PLP-Dependent Enzymes

A Functional Genomics Approach

A recent study demonstrated that about 1.5% of all genes inmost prokaryotic genomes encode PLP-dependent enzymesand that 4% of all enzyme activities classified by the EnzymeCommission depend on PLP.92 Furthermore, there is no assign-ment of catalytic activity for about 20% of the putative PLP-dependent enzymes encoded by the human genome, andone-third of all PLP-dependent enzymes classified by theEnzyme Commission are still uncharacterized in terms ofsequence.93 These numbers are enough to evidence the lack ofinformation regarding the family of PLP-dependent enzymes,and, on the other side, how much work is needed before tofully exploit the wealth of information contained in thegenome sequences. Some questions that have arisen are “whatis the minimal set of such enzymes that is required by a free-living organism? How different are the number and variety of


285

these enzymes in microorganisms and higher eukaryotes? Doesgenomic analysis suggest the existence of novel, as yet unclas-sified, PLP-dependent enzymes?”92 Indeed, “the occurrence ofcatalytic promiscuity and loose substrate specificity impliesthat an organism may have more PLP-dependent activitiesthan it has genes encoding PLP-dependent enzymes.”92

A Family With a Growing Number of Drug Targets

In spite of the high representativeness of PLP-dependentenzymes on the overall enzymatic activities, only about 20 ofthem have been so far identified as potential targets for thera-peutic agents in human diseases or for herbicides. Even less arethe PLP-dependent enzymes for which drugs have been devel-oped. These are DOPA decarboxylase in Parkinson’s disease,GABA aminotransferase in epilepsy, serine hydroxymethyl-transferase in tumors and malaria, ornithine decarboxylase inAfrican sleeping sickness and, potentially, in tumors, alanineracemase as an antibiotic, and human cytosolic branched-chain aminotransferase in pathological states associated to theGABA/glutamate equilibrium concentrations. This verylimited number is associated with the lack of knowledge of therole played by PLP-dependent enzymes in a variety of biolog-ical processes, and in pathological states. As a result of the func-tional genomics approach, the increased availability of purifiedPLP-dependent enzymes, and the understanding of cellularevents, more potential drug targets have been and will be iden-tified. Representative examples are (i) cystathionine beta-synthase and cystathionine gamma-lyase, involved in the production of H2S, a compound recently found to act as a neu-romodulator;94,95 (ii) the exploitation of mAb-allinase conju-gates for in situ production of the cytotoxic allicin;96 (iii) theselective inhibition of 3-hydroxykynurenine transaminase inAnopheles gambiae as therapeutic strategy for blocking malariatransmission, taking advantage of the absence of this enzyme

in humans;97 and (iv) the PLP-dependent enzymes involved inkynurenine formation and degradation.98,99

A Model Protein Family for Comparative Folding Studies

PLP-dependent enzymes belong to five distinct structuralclasses.3 They share an oligomeric assembly and the binding of the coenzyme, which is usually localized at the interfacebetween two domains, and, in several cases, at the subunitinterface. Detailed investigations of the folding mechanism arelimited due to inherent difficulties of characterizing multisub-unit protein stability, where unfolding and refolding mightdepend on subunit dissociation–association, coenzyme uptakeand release, complete or partial reversibility, oxidative or reduc-ing conditions, and speed of concurrent processes as cis-transproline isomerization. A comparative folding–unfolding studyon PLP-dependent enzymes that share the same structural fea-tures (same fold type), but exhibit sequence homology evenlower than 30%, would be extremely valuable for the under-standing of the key residues that control both the catalyticfunction and the structural organization. A similar study canbe carried out comparing folding mechanisms of enzymesbelonging to different fold types. This effort would require a coordinated network of several investigators on PLP-dependent enzymes.

We thank the Ministry of University and Research for thecontinuous financial support (COFIN 1999, 2001, 2003,and 2005).

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Table 2. Specific activities for reactions catalyzed by tryptophan synthase (TS), tyrosine phenol lyase (TPL), and tryptophan indole lyase(Trpase) in solution and encapsulated in silica gels.

Catalytic activity Catalytic activityEnzyme Reaction Substrate (Umg−1)-solution (Umg−1)-gel

TSa β-replacement L-serine 880 ± 10 95 ± 5TSa β-replacement β-Cl-alanine 158 ± 9 53 ± 1TSa β-elimination L-serine 20 ± 1 8.4 ± 0.1TSa β-elimination β-Cl-alanine 25 ± 1 20 ± 1TPLb α-β-elimination S-(o-Nitrophenyl)-L-cysteine 4.1 ± 0.1 0.49 ± 0.07TPLb α-β-elimination S-methyl-L-cysteine 0.21 ± 0.01 ∼0Trpaseb α-β-elimination S-(o-Nitrophenyl)-L-cysteine 15.2 ± 0.5 1.8 ± 0.2Trpaseb α-β-elimination S-methyl-L-cysteine 3.7 ± 0.5 0.62 ± 0.10

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