Biochemical Characterization of the THIN-B Metallo-{beta}-Lactamase of Janthinobacterium lividum

The THIN-B metallo-ß-lactamase, a subclass B3 enzymeproduced by the environmental species Janthinobacterium lividum,was overproduced in Escherichia coli by means of a T7-basedexpression system. The enzyme was purified (>95%) by twoion-exchange chromatography steps and subjected to biochemicalanalysis. The native THIN-B enzyme is a monomeric protein of31 kDa. It exhibits the highest catalytic efficiencies withcarbapenem substrates and cephalosporins, except for cephaloridine,which acts as a poor inactivator. Individual rate constantsfor inactivation by chelators were measured, suggesting thatinactivation occurred by a mechanism involving formation ofa ternary complex.

INTRODUCTION

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Introduction

Metallo-ß-lactamases (MBLs) are the focus of increasinginvestigation both as resistance determinants and as model enzymes.As resistance determinants their relevance depends on theirfunctional features (broad substrate specificity, efficientcarbapenemase activity, resistance to the so-called "mechanism-based"ß-lactamase inactivators) and on the recent emergenceof MBLs encoded by genes associated with mobile DNA among majorbacterial pathogens (19, 21, 23, 24). The interest in MBLs asmodel enzymes arises from the as yet superficial understandingof their catalytic mechanism and structure-function relationships,which could be essential to the development of new ß-lactamsand enzyme inhibitors. On the other hand, the MBL fold is conservedwithin a large protein superfamily that includes a growing numberof proteins which do not hydrolyze ß-lactams (2, 6,8).

MBLs belong to molecular class B (1) and constitute a familyof very diverse enzymes. Based on structural relatedness, theycan be grouped into three different subclasses: B1, B2, andB3 (14, 23). Subclass B3, originally represented by the L1 enzymefrom Stenotrophomonas maltophilia (4, 26), has recently expandedto include several enzymes from primarily environmental bacteria(FEZ from Legionella gormanii [5], GOB from Chryseobacteriummeningosepticum [3], THIN-B from Janthinobacterium lividum [25],and CAU from Caulobacter crescentus [9]), some of which canoccasionally behave as opportunistic pathogens. The MBLs ofsubclass B3 are highly divergent from those of subclass B1 atthe sequence level (14) and, although they retain a three-dimensionalfold that is similar overall, exhibit an organization of themetal-binding sites that differs significantly from that ofenzymes of subclass B1 (15, 28).

The THIN-B enzyme from J. lividum was identified following anenvironmental screening of MBL-producing bacteria (25) and currentlyis the only known MBL from a member of the ß-Proteobacteriaclass. Compared to the other enzymes of subclass B3, THIN-Bis quite divergent and exhibits some unique structural features,including a larger size and a higher number of cysteine residues(25). However, the biochemical properties of this enzyme havenot been investigated.

In this paper we describe a system for overproduction of theTHIN-B enzyme in Escherichia coli, the protocol for purificationof the recombinant protein, and the biochemical and kineticcharacterization of THIN-B.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. E. coli XL-1 Blue (Stratagene, Inc., La Jolla, Calif.) was usedas a host for recombinant plasmids. E. coli strains BL21(DE3)(Stratagene), BL21-SI (Invitrogen, Carlsbad, Calif.), and MCT236(DE3){E. coli CGSC6159 [lacI^q rrnB_T14 ${Delta}$ lacZ_WJ16 hsdR514 ${Delta}$ araBA-D_AH33 ${Delta}$ rhaBAD_LD78 ${Delta}$ pyrC ( ${lambda}$ cIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)]}(constructed in our laboratory) were used as hosts for T7 promoter-basedexpression plasmids for bla_THIN-B overexpression experiments.

Media and culture conditions. Bacteria were always grown aerobically at 37°C unless otherwisespecified. Luria-Bertani medium (27) was routinely used forthe propagation of E. coli strains. SB medium (20 g of yeastextract/liter, 35 g of tryptone/liter, and 5 g of NaCl/liter;buffered with 50 mM sodium phosphate buffer [pH 7.0]) was usedin overexpression experiments with BL21(DE3) and MCT236(DE3)strains. LBON (5 g of yeast extract/liter and 10 g of tryptone/liter)was used in overexpression experiments with BL21-SI.

Recombinant DNA methodologies. The open reading frame encoding THIN-B was amplified by PCRusing primers THIN-B-EXP/f (5'-CAT ATG ACA CTA TTG GCG AAG TTGATG CTG), which added an NdeI linker (boldfaced) to the 5' end,and THIN-B-EXP/r (5'-GGA TCC TAG TGC GCG TGC TGG G), which addeda BamHI linker (boldfaced) to the 3' end. PCR was performedin a volume of 50 µl, with 3.5 U of the Expand High FidelityPCR system (Roche Biochemicals, Mannheim, Germany) in the bufferprovided by the manufacturer, 200 µM deoxynucleoside triphosphates,50 pmol of each primer, and 10 ng of plasmid pBCIRO-K (25) asthe template for the bla_THIN-B gene. The following cycling conditionswere used: initial denaturation at 94°C for 3 min; denaturationat 94°C for 60 s, annealing at 58°C for 60 s, and extensionat 72°C for 90 s, repeated for 30 cycles; and a final extensionstep at 72°C for 10 min. The amplified DNA was cloned intothe SmaI site of plasmid pUC-18 by using the Sure Cloning kit(Amersham Biosciences, Uppsala, Sweden), yielding the recombinantplasmid pUC-CIRO. Finally, the 0.95-kb NdeI-BamHI fragment ofpUC-CIRO, containing the bla_THIN-B gene, was subcloned intothe pET-9a expression vector to produce the recombinant plasmidpET-THIN-B. The cloned bla_THIN-B gene was sequenced to ruleout the presence of any PCR-generated mutations.

Expression experiments. THIN-B production was tested by using three different expressionsystems, obtained by transformation of E. coli BL21(DE3), BL21-SI,or MCT236(DE3) with pET-THIN-B. With each system MBL productionwas monitored, over a 24 h time course, in both supernatantsand cell extracts of cultures growing in SB medium containing50 µg of kanamycin/ml at either 25 or 37°C. Individualcultures were incubated until the A₆₀₀ reached 0.8 and thenwere split into two identical flasks, to one of which was added1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) [forthe BL21(DE3) and MCT236(DE3) hosts] or 0.3 M NaCl (for theBL21-SI host). Aliquots (1 ml) were sampled at different timesand centrifuged (12,000 x g, 5 min, 4°C), and the culturesupernatant was stored at 4°C. The bacterial pellet wasresuspended in 1 ml of 10 mM HEPES-NaOH (pH 7.5) supplementedwith 50 µM ZnSO₄ and was disrupted by sonication (5 cycles,for 20 s each cycle, at 45 W) using a B. Braun (Melsungen, Germany)Labsonic L sonicator. The supernatant obtained after centrifugationat 10,000 x g for 15 min, to remove cell debris, representedthe cell extract. MBL activities in supernatants and cell extractswere determined spectrophotometrically at 30°C by using150 µM imipenem as the substrate (wavelength, 300 nm; ${Delta}$ ${varepsilon}$ , –9,000 M^–1 · cm^–1) in 10 mM HEPES-NaOHbuffer (pH 7.5) (20). The reaction volume was 500 µl.

Purification of the THIN-B enzyme. The THIN-B MBL was purified from a culture of E. coli MCT236(DE3)(pET-THIN-B)grown in 0.5 liter of SB medium at 25°C. The culture wasinduced with 1 mM IPTG when the A₆₀₀ was equal to 0.8. Cellswere collected, 24 h after induction, by centrifugation (10,000x g, 15 min, 4°C), resuspended in 20 ml of 20 mM ethanolamine-NaOHbuffer (pH 9.8) containing 1 mM MgCl₂ and 50 µM ZnSO₄,and disrupted by sonication as described above. Cellular debriswas removed by centrifugation (12,000 x g, 60 min, 4°C).The cleared supernatant was desalted by using a HiPrep 26/10desalting column (Amersham Biosciences) and loaded (flow rate,2 ml/min) onto an HR column (1.6 by 5 cm) packed with 10 mlof Source 15Q (Amersham Biosciences), equilibrated with 20 mMethanolamine-NaOH buffer (pH 9.8) (buffer A). Proteins wereeluted (flow rate, 4 ml/min) by using a linear gradient of NaCl(0 to 0.2 M, in 40 ml) in buffer A. Fractions exhibiting imipenemaseactivity were pooled and concentrated by ultrafiltration usinga Centriprep YM10 filter unit (Millipore, Bedford, Mass.), andthe buffer was changed to 50 mM sodium acetate buffer (pH 5.0)(buffer B) by using a PD-10 disposable column (Amersham Biosciences).The sample was then loaded (flow rate, 1 ml/min) onto a ResourceS column (6.4 by 30 mm; Amersham Biosciences) preequilibratedwith buffer B and was eluted by using a linear NaCl gradientin buffer B (0 to 1 M, in 20 ml). Fractions exhibiting the highestimipenemase activity were pooled, and the buffer was changedto 50 mM HEPES-NaOH (pH 7.5) containing 50 µM ZnSO₄ (HBZbuffer), as above. The purified enzyme was stored in HBZ bufferat –80°C until use. During purification, imipenemaseactivity was assayed as described for expression experiments(see preceding section).

Protein analysis techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed by the method of Laemmli (18) by using a 12% acrylamideconcentration in the resolving gel. After electrophoresis, proteinbands were stained with Coomassie brilliant blue. Analyticalisoelectric focusing (IEF) of the purified protein and zymographicdetection of ß-lactamase activity were carried outas described previously (21). The molecular mass of native THIN-Bwas estimated by size exclusion chromatography using a Superdex75 HR 10/30 column (Amersham Biosciences) as described previously(9, 10). The column was calibrated by using a mixture of bovineserum albumin (0.2 mg), ovalbumin (0.2 mg), chymotrypsinogenA (0.2 mg), and RNase A (0.5 mg). The protein concentrationin solution was determined by using a commercial kit (Bio-Rad[Richmond, Calif.] protein assay) with bovine serum albuminas the standard. The BBL numbering scheme (14) is used throughoutthis paper.

Mass spectrometry. The mass of the purified THIN-B protein was measured by usingan Ettan MALDI-TOF Pro mass spectrometer (Amersham Biosciences).The enzyme preparation had previously been desalted by usinga ZipTip C4 (Millipore Corporation). Protein identificationby mass spectrometry was achieved by peptide mass fingerprinting(PMF) (17). Briefly, the purified THIN-B protein was digestedin 50 mM NH₄HCO₃ buffer (pH 7.0) by using trypsin (Promega,Madison, Wisc.) at a final protease/protein ratio of 1:50; thereaction took place at 37°C for 1 h. The mass spectra ofthe peptides in the mixture were acquired by using an EttanMALDI-TOF Pro mass spectrometer (Amersham Biosciences), andthe experimental data were compared to theoretical PMFs calculatedfor all the sequences in DNA and protein sequence databasesby using the database search engine MASCOT, available online(http://www.matrixscience.com).

Determination of kinetic parameters. The steady-state kinetic parameters for ß-lactam hydrolysiswere measured in HBZ buffer at 30°C as described previously(9, 10). Competition experiments with aztreonam were carriedout by using 200 µM imipenem as the reporter substrateand aztreonam concentrations up to 1 mM. Inactivation rates(k_i) with divalent metal chelators were determined as describedpreviously (10) in 50 mM HEPES-NaOH buffer (pH 7.5) (HB buffer)at 30°C by using 1 mM meropenem as the reporter substrate.Data were analyzed as described previously (16) according tothe following scheme:

where E ·Zn is the metalloenzyme, E is the inactive apoenzyme, C is themetal chelator, Zn · C is the metal-chelator complex,E · Zn · C is the ternary metal-enzyme-chelatorcomplex, K is the dissociation constant for the ternary complex,k₊₂ is the rate constant for dissociation of the ternary complex,and k_–₂ is the second-order rate constant for formationof the ternary complex. The individual constants K, k₊₂, andk_–₂' (the pseudo-first-order rate constant for formationof the ternary complex from the apoenzyme and the metal-chelatorcomplex) were calculated by use of a nonlinear regression byfitting the equation to the experimental data, where k_–₂' is k_–₂ · [Zn · C]and K' is , where K_m^S is the K_m for the reportersubstrate (16). The enzyme concentration in kinetic assays rangedbetween 3.5 and 95 nM.

Determination of free sulfhydryl groups in the protein. The free sulfhydryl groups were titrated in unfolded purifiedprotein samples (after addition of 6 M guanidinium chloride),in either unreduced or reduced forms, by using Ellman's assay,as described previously (7). The reduced form was obtained afterincubation of the protein in the presence of 10 mM dithiothreitolfor 1 h at 20°C and subsequent removal of the excess reducerby desalting the sample by use of a PD-10 column (Amersham Biosciences).The final enzyme concentration in the assay was 0.1 mg/ml.

RESULTS AND DISCUSSION

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Results and Discussion

THIN-B production in E. coli. The bla_THIN-B gene was cloned in the T7-based expression vectorpET-9a, and enzyme production was tested by using three differentE. coli hosts producing T7 RNA polymerase. Pilot expressionexperiments, in which cultures were grown at 37°C, yieldedpoor ß-lactamase activity both in culture supernatantsand in cell extracts (specific imipenemase activity was alwayslower than 100 nmol/min/ml) (data not shown). Lowering the incubationtemperature to 25°C significantly increased the ß-lactamaseyield (approximately 3- to 14-fold, depending on the host).Comparative analysis of ß-lactamase production inthe three different expression systems showed that additionof the inducer generally improved the yield of recombinant proteinand that the highest activity was observed in the cell extractsafter 24 h (Fig. 1). ß-Lactamase activity was alsomeasured in the culture supernatants at different times, butno trace of enzyme was found in the corresponding samples, exceptwith the BL-21-SI-based system, for which significant cell lysiswas observed when the inducer (NaCl at 0.3 M) was added to theculture (data not shown). The highest yield was obtained withMCT236(DE3)(pET-THIN-B) after induction with 1 mM IPTG, andthis system was adopted for larger-scale THIN-B production andpurification.

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FIG. 1. Time-dependent production of the THIN-B enzyme in cell lysates with different expression systems. E. coli BL21(DE3)(pET-THIN-B), white; E. coli BL21-SI(pET-THIN-B), light grey; E. coli MCT236(DE3)(pET-THIN-B), dark grey. Hatched bars represent samples from cultures to which an inducer (1 mM IPTG, or 0.3 M NaCl for BL21-SI) was added.

THIN-B purification. THIN-B was purified from a cell extract of E. coli MCT236(DE3)(pET-THIN-B),obtained from a 0.5-liter culture induced with IPTG and grownat 25°C for 24 h, by means of two ion-exchange chromatographysteps, an anion exchange at pH 9.8 followed by a cation exchangeat pH 5.0. No detectable changes in enzyme activity were registeredwithin 2 h of exposure at such pH values. A summary of the purificationprocess is shown in Table 1. Higher activity was observed afterdesalting of the crude extract, presumably due to removal oflow-molecular-weight inhibitors whose nature was not specificallyinvestigated. The yield of purified protein was approximately30 mg per liter of culture. The purity of the protein preparationwas estimated to be >95%, according to SDS-PAGE analysis(data not shown). Recovery was relatively low (30%), mainlybecause, after the cation-exchange step, only the purest fractionswere retained.

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TABLE 1. Summary of a typical purification procedure of the THIN-B enzyme from E. coli MCT236(DE3)(pET-THIN-B)

Matrix-assisted laser desorption ionization—time-of-flight(MALDI-TOF) mass spectrometry yielded an average mass of 30,915± 45 Da for the purified THIN-B protein. The mass spectrumof the enzyme tryptic digest, obtained by using a MALDI-TOFspectrometer, yielded nine peaks, all at m/z ratios in agreementwith those predicted by computing analysis, and covering 43%of the protein sequence (Table 2). This confirmed the authenticityof the enzyme preparation.

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TABLE 2. MALDI-TOF mass spectrum data obtained for the THIN-B protein preparation after digestion with trypsin^a

Structural features of THIN-B. Analytical IEF of the purified protein preparation revealeda pI of 6.3 ± 0.3 (data not shown), in good agreementwith the calculated value for the mature protein (pI 6.22).

Size exclusion chromatography yielded a molecular mass of 32± 2 kDa, indicating that, under the experimental conditionsadopted, the native enzyme is monomeric, unlike L1 but likemost other MBLs (3, 4, 9, 22). The absence of oligomerizationin THIN-B, despite its longer N-terminal portion, is consistentwith the absence of the residues that are known to be involvedin oligomerization of L1 (28). In particular, the methionineresidue at position 175, which is responsible for hydrophobicintersubunit interactions involved in formation of the L1 dimer,is replaced in THIN-B by a glycine whose side chain lacks anypossibility of interacting with the hydrophobic pocket of anothersubunit. In addition, of the three hydrophobic residues (Leu154,Pro198, Tyr236) that form the L1 hydrophobic pocket where Met175is accommodated, only Pro198 is conserved in THIN-B (Fig. 2).

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FIG. 2. Amino acid sequence alignment of the THIN-B MBL in comparison with the other subclass B3 enzymes (L1 from S. maltophilia IID1275 [29], FEZ-1 from L. gormanii ATCC 33297 [5], GOB-1 from Chryseobacterium meningosepticum PINT [3], and CAU-1 from Caulobacter crescentus DSM4727 [9]). Residues that are identical in all sequences are boldfaced. The structural elements of L1 are indicated above the sequences ( ${alpha}$ and 3₁₀, helices; ß, strands). Cysteine residues are shaded.

The THIN-B enzyme contains six cysteine residues, remarkablymore than are found in the mature GOB-1 (one cysteine residue),L1 or CAU-1 (two cysteine residues), and FEZ-1 (three cysteineresidues) proteins (Fig. 2). Determination of the free sulfhydrylgroups yielded free cysteine/enzyme ratios of 2.1 ± 0.3and 5.6 ± 0.4 for the unreduced and reduced proteins,respectively. This suggested that, in THIN-B, four cysteinesmight be involved in the formation of two disulfide bridges.One of these is likely formed by the Cys256 and Cys290 residues,which in L1 and FEZ-1 are known to form a disulfide bridge thatlinks the last strand of the ß-sheet in the C-terminaldomain with the terminal ${alpha}$ -helix (15, 28), and which are conservedin THIN-B. The other disulfide bridge would most likely involveresidues 208 and 213, which are located at the extremities ofthe ß11 and ß12 strands, respectively (15).Alternatively, the disulfide bridge could involve the two cysteineresidues at positions 32 and 35, which are located close tothe N terminus of the protein (Fig. 2).

Another notable feature of THIN-B, compared to other enzymesof subclass B3, is its larger protein size (Fig. 2). THIN-Bis 29 and 33 amino acids longer, respectively, than L1 and FEZ-1,the two subclass B3 enzymes whose three-dimensional structureshave been solved. Sequence comparison and consideration of structuraldata indicate that most of those extra amino acids are likelyto be part of a longer N-terminal coil and of a longer C-terminalß-helix, with no important modifications of internalelements (Fig. 2).

Kinetic properties of the THIN-B enzyme. Determination of the kinetic parameters of THIN-B with severalsubstrates representative of different ß-lactam families,including penicillins, narrow- to expanded-spectrum cephems,carbapenems, and aztreonam, revealed that all the compoundstested, except aztreonam, were consistently hydrolyzed by theenzyme, although with a remarkable variability of k_cat/K_m ratios(from 8 x 10³ to 5 x 10⁶ M^–1 · s^–1) (Table3). The highest catalytic efficiencies (k_cat/K_m ratios, >10⁶M^–1 · s^–1) were observed with carbapenems,cefuroxime, and cefotaxime, while cefepime appeared to be apoor substrate. Comparison of K_m values indicated a higher apparentaffinity for carbapenems and cephalosporins (except cefepime)than for penicillins. The highest turnover rate was observedwith ampicillin (k_cat, 480 s^–1), a substrate for whichthe enzyme also exhibited the highest K_m (1.3 mM), resultingin a relatively low catalytic efficiency compared to those ofexpanded- and broad-spectrum cephalosporins and carbapenems(Table 3). Aztreonam was not hydrolyzed and did not interactwith the enzyme (Table 3), as has also been observed with otherMBLs (3, 9-13, 20, 22, 23). With cephaloridine (at a substrateconcentration of 100 µM), the hydrolysis kinetics presenteda notable though slow inactivation pattern until an apparentsteady state was reached (rate, 650 µmol/min/mg of enzyme)(Fig. 3). Although the inactivation mechanism has not been furtherinvestigated, the most reliable hypothesis possibly explaininga similar behavior could be the existence of two enzyme-substratecomplex forms with significantly different turnover rates, leadingto accumulation of the enzyme-substrate complex with minor activity.It would be interesting to further investigate this point bymass spectrometry experiments.

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TABLE 3. Kinetic parameters of the purified THIN-B metallo-ß-lactamase and comparison with catalytic efficiencies of other subclass B3 enzymes^a

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FIG. 3. Time course for cephaloridine hydrolysis in the presence of 13 nM THIN-B. A steady state was reached after approximately 10 min. Cephaloridine was normally hydrolyzed when 8 nM VIM-2 (10) was added to the reaction mixture, as indicated by the arrow.

THIN-B exhibits a broad substrate profile, similar to that ofother MBLs of subclass B3 (3, 9, 11, 12, 22). Comparison ofthe kinetic parameters of these enzymes reveals both commonfeatures and functional differences. The carbapenemase activitytends to be quite efficient in all cases, with an overall preferencefor meropenem, while cefepime tends to behave as a relativelypoor substrate in most cases. On the other hand, large differencesin catalytic efficiency can be observed with penicillins andsome cephems (Table 3). The slow inactivation of THIN-B by cephaloridineis certainly an uncommon feature.

Inactivation of THIN-B by metal chelators. The THIN-B enzyme was efficiently inactivated by EDTA, o-phenanthroline,and dipicolinic acid, allowing the determination of pseudo-first-orderinactivation rates (k_i).

With EDTA and dipicolinic acid, the inactivation rates variedwith the inactivator concentration, following a hyperbolic dependence,and individual inactivation constants (K, k₊₂, k_–₂') couldbe calculated (Fig. 4; Table 4). This behavior suggests thatthe inactivator does not act by simply scavenging the free metalfrom the buffer but that a ternary enzyme-metal-inactivatorcomplex is formed during inactivation. No similar behavior hasbeen observed for other enzymes of subclass B3 (9, 22), andTHIN-B represents the first example of a subclass B3 enzymefor which formation of the ternary complex could be hypothesized.

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FIG. 4. Dependence of the pseudo-first-order inactivation rate (k_i) of THIN-B on the concentration of the metal chelator EDTA (a) or dipicolinic acid (b). The line in each graph represents the best fit resulting from the nonlinear regression used to calculate individual inactivation parameters (K, k₊₂, and k_–₂').

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TABLE 4. Inactivation parameters for the THIN-B metallo-ß-lactamase with various chelating agents^a

With o-phenanthroline, the measured inactivation rate [k_i, (1.22± 0.06) x 10^–2 s^–1] was independent of theinactivator concentration (in the range of 50 to 500 µM)and corresponded to the k₊₂ constant, indicating that formationof the ternary complex was rather efficient (K, <50 µM)(Table 4).

The inactivation efficiencies of chelators were generally high,with values similar to those observed for subclass B1 enzymes(10, 13, 20). Interestingly, with THIN-B, EDTA was a betterinactivator than dipicolinic acid or o-phenanthroline (Table4), at variance from what has generally been observed for MBLs(9, 10, 13, 16, 20, 22).

Concluding remarks. Subclass B3 of MBLs, which until recently included only theL1 enzyme from S. maltophilia, currently includes several membersof notable structural and functional diversity. Although producedby a nonpathogenic bacterium, the THIN-B enzyme exhibits somepeculiar structural and functional features and could be aninteresting model for further structural studies. Moreover,the distribution of MBLs among an increasing number of bacterialspecies typically associated with environmental niches thatmight act as a reservoir for efficient resistance determinantsmight offer an important clue for MBL evolution.

ACKNOWLEDGMENTS

This work was supported by the European Research Network onmetallo-ß-lactamases (contract HPRN-CT-2002-00264)and by a grant from the Italian Ministero dell'Istruzione, dell'Universitàe della Ricerca (PRIN 2003). J.-D.D. is funded by the "FondsNational de la Recherche Scientifique" of Belgium as a "CollaborateurScientifique F.N.R.S."

FOOTNOTES

* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Università di Siena, Policlinico Le Scotte, 53100 Siena, Italy. Phone: 39 0577 233327. Fax: 39 0577 233325. E-mail: rossolini{at}unisi.it.

Present address: Laboratoire d'Enzymologie et Centre d'Ingénieriedes Protéines, Institut de Chimie, Universitéde Liège, B-4000 Liège, Belgium.

REFERENCES

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