Aspartate aminotransferase from the Antarctic bacteriumPseudoalteromonas haloplanktis TAC 125Cloning, expression, properties, and molecular modelling

Leila Birolo1,*, M. Luisa Tutino1,*, Bianca Fontanella1, Charles Gerday2, Katia Mainolfi1, Stefano Pascarella3,Giovanni Sannia1, Floriana Vinci1 and Gennaro Marino1

1Dipartimento di Chimica Organica e Biologica, UniversitaÁ di Napoli Federico II, Italy; 2Laboratory of Biochemistry,

Institut de Chimie B6a au Sart-Tilman, Liege, Belgium; 3Dipartimento di Scienze Biochimiche A. Rossi Fanelli, UniversitaÁ di Roma La Sapienza,

Italy

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis

TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was

characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which

is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 8C(half-life � 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for

PhAspAT activity is < 64 8C, which is some 11 8C below that of EcAspAT. The protein thermal stability was

investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested

that a first structural transition occurs at < 50 8C for PhAspAT. These results agree with the expected

thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for

enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by

several extracellular psychrophilic enzymes, both kcat and kcat /Km of PhAspAT are significantly lower than those

of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class

of endocellular enzymes to a cold environment may have only made them less stable and not more efficient.

The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature.

However, binding of maleate and 2-methyl-l-aspartate, which both inhibit the initial steps of catalysis, does not

change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the

steps of the catalytic mechanism after the formation of the external aldimine.

A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs.

Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing

can be observed.

Keywords: aspartate aminotransferase; cold adaptation; psychrophile.

The conformational stability of most globular proteins issurprisingly low, usually not exceeding 60 kJ´mol21 [1,2],which corresponds to a few non-covalent interactions (hydrogenbonds, salt bridges and hydrophobic interactions) out ofhundreds that contribute to the formation of a well-defined3D protein structure. The biological significance of thisobservation may depend on the functional role of proteins: acompromise between rigidity and flexibility of the polypeptidechain is absolutely required to allow the protein to function.This compromise reflects the basic need for biologicalmolecules to adapt to `extreme' growth conditions, as evolution

seems to have forced structural changes towards optimizationof structure/function relationships rather than maximumstability [2].

Only recently has increased effort been devoted to under-standing the molecular basis of protein adaptation to cold(reviewed in [3±5]). Life at low temperature generally requiresmolecular modifications that are not just the opposite of thoseadopted by thermophilic organisms. `Heat-adapted' proteinsindeed face the problem of stability at high temperature, butpsychrophilic proteins have to cope with the reduction inreaction rates at temperatures close to 0 8C in order to maintainadequate metabolic fluxes. A more flexible structure seems thesimplest strategy of enzyme adaptation to catalysis at lowtemperature in order to undergo the rapid and reversibleconformational modifications that are required by the catalyticcycle. In fact, although only a few psychrophilic proteins,mainly extracellular, have been studied so far, it seems thatmost `cold-adapted' enzymes tend to increase flexibility ofthe structure by weakening intramolecular interactions andincreasing interactions with the solvent [3].

An interesting paradigm for studies focusing on structure/function/stability relationships is aspartate aminotransferase

Eur. J. Biochem. 267, 2790±2802 (2000) q FEBS 2000

Correspondence to G. Marino, Dipartimento di Chimica Organica e

Biologica, UniversitaÁ di Napoli Federico II, Via Mezzocannone 16,

I-80134 Napoli, Italy. Fax: 1 39 081 7041202, Tel.: 1 39 081 7041252,

E-mail: gmarino@unina.it

Abbreviations: AspAT, aspartate aminotransferase; CSA, l-cysteine

sulphinate; PhaspC, gene coding for aspartate aminotransferase from

Pseudoalteromonas haloplanktis TAC 125.

Enzyme: aspartate aminotransferase (EC 2.6.1.1)

* These authors contributed equally to the paper.

(Received 14 February 2000, accepted 13 March 2000)

(AspAT), a ubiquitous enzyme, purified and characterized frommany sources, the 3D structures of which have been solvedin mammals (pig cytosolic) [6], birds (chicken mitochondrial)[7±9] and bacteria (Escherichia coli ) [10,11]. A comparativeanalysis of the hyperthermophilic AspAT from the ArchaeonSulfolobus solfataricus with mesophilic counterparts isolatedfrom pig heart and E. coli suggests that the quaternary structureof the thermophilic protein is stabilized by a network ofhydrophobic interactions, while new hydrogen bonds and ionpairs contribute to the stabilization of the tertiary structure ofthe single subunit [12].

To extend these studies to cold-loving organisms, we investi-gated the molecular adaptation of AspAT from the psychro-philic Antarctic bacterium Pseudoalteromonas haloplanktisTAC 125 (formerly Moraxella TAC 125).

We report cloning and expression of the gene codingfor AspAT from Ps. haloplanktis TAC 125 (PhAspAT), andcharacterization of the recombinant enzyme, including use of amolecular model of the protein.

E X P E R I M E N T A L P R O C E D U R E S

Bacterial sources and plasmids

The bacterial isolate TAC 125 (C. Gerday collection), formerlyreported as Moraxella TAC 125, was classified by DSMZ,Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, Munich, Germany as Ps. haloplanktis TAC 125. Thisgram-negative strain was collected in 1992 from sea water nearthe French Antarctic Station Dumont d'Urville (60840 0S;40801 0E). E. coli strain TY103 [Dlacpro, supE, thi, strA,sbcB15, endA, hsdR4, F 0(traD36, proAB1, lacI qZDM15),aspC::kan, tyrB::cat, recA::tet] [13] and plasmid pKDHE19[14] were gifts from P. Christen, Biochemiches Institut derUniversitaÈt ZuÈrich, Switzerland. E. coli strain TOP F 010[F 0{lacI q Tn10(TetR)} mcrA, D(mrr-hsdRMS-mcrBC), F80lacZDM15, DlacX74, deoR, recA1, araD139, D(ara-leu)7697,galU, galK, l-, rspL, endA1 nupG ] was the host strain used forlibrary construction.

Enzyme assays

During purification, enzyme activity was routinely measured bythe conventional malate dehydrogenase-based assay [15], theconditions being 25 8C in 0.1 m Hepes (sodium salt), pH 8.0,containing 18 mm aspartate,7 mm 2-oxoglutarate, 0.15 mmNADH and malate dehydrogenase (2.5 mg´mL21).

Partial purification of PhAspAT and N-terminal sequencedetermination

Ps. haloplanktis TAC 125 was grown to saturation at 4 8C inTYP broth (16 g´L21 yeast extract, 16 g´L21 bacto-tryptone,10 g´L21 sea salts at pH 7.5). Cells were recovered bycentrifugation at 3500 g, 4 8C for 15 min; the cell pellet wassuspended (1 mL per g wet cells) in lysis buffer (50 mm Tris/HCl, 10 mm EDTA, 100 mm NaCl pH 8.0, 0.1 mm pyridox-amineP, 5 mm 2-oxoglutarate, 1 mm dithiothreitol, 135 mmphenylmethanesulfonyl fluoride) and then disrupted by abrasivetreatment. The homogenate was centrifuged for 25 min at60 000 g, 4 8C, and the clear cytosolic extract loaded on aSuperdex 200 prep grade 35/600 column (Pharmacia BiotechInc.) equilibrated in 50 mm Tris/HCl/0.15 m NaCl, pH 7.0;fractions containing AspAT activity were equilibrated in 50 mmtriethanolamine/HCl, pH 6.0, loaded on a Mono Q HR 5/5

column (Pharmacia) equilibrated in the same buffer, and theneluted with a linear gradient of 0±0.3 m NaCl (65 mL).Proteins in the AspAT active fractions were separated bySDS/PAGE and electroblotted on a poly(vinylidene difluoride)membrane. The N-terminal sequence of PhAspAT was deter-mined directly from the excised band by stepwise Edmandegradation with a Perkin-Elmer Applied Biosystem 477Apulsed liquid protein sequencer equipped with 120A HPLCapparatus for the on-line phenylthiohydantoin-amino-acididentification.

Ps. haloplanktis TAC 125 genomic DNA purification andlibrary construction

Genomic DNA was isolated from the bacterial pellet of 250 mLof Ps. haloplanktis TAC 125 culture at saturation. Cells weresuspended in 8 mL of LETS buffer (LiCl 100 mm, EDTA10 mm, Tris/HCl 10 mm, pH 7.8, SDS 1%) and extracted threetimes with an equal volume of phenol/chloroform/isoamylicalcohol (25 : 24 : 1, by vol.), the first time in the presence of4 g sand. Nucleic acids were then recovered from the aqueousphase by ethanol precipitation. RNA and residual proteins weredigested in a 30-min treatment with RNase A (150 mg´mL21)and proteinase K (50 mg´mL21) at 37 8C. Chromosomal DNAwas digested with HindIII or PstI and then ligated into theHindIII-digested or PstI-digested pGEM-4z vector (Promega)without prior fractionation. The ligated DNA fragments weretransferred into E. coli TOP F 010 by electroporation, andtransformants were selected on Luria±Bertani agar platescontaining 100 mg ampicillin´mL21.

Cloning of Ps. haloplanktis TAC 125 gene coding for PhAspAT

A DNA fragment of 1047 bp of the gene coding for thePs. haloplanktis TAC 125 AspAT (PhaspC ) was amplified byPCR on genomic DNA. The N-terminal sequence of the proteinwas used to design a degenerated oligonucleotide (oligo 5 0: 5 0-ATGTTYTCNGTNYTWAARC, Y � T, C; N � T, C, G, A;W �A, T; R � A, G) which was used in combination with theoligo 3 0 (5 0-RAANGARAACATNCCRTTYTG) designed on ahighly conserved region in the amino-acid sequences of severalmicrobial AspATs (see Fig. 1). A mixture containing 150 nggenomic PstI-hydrolysed DNA, 1 nmol of each oligonucleotideprimer, 1.8 mm MgCl2, 50 mm KCl, 20 mm Tris/HCl pH 8.3,0.1% gelatin, 200 mm dNTP in a final volume of 70 mL, wasincubated at 95 8C for 10 min, after which 2.5 units Taq DNApolymerase was added. A total of 29 cycles of amplificationconsisting of 1 min at 95 8C, 90 s at 52 8C and 2 min at 72 8Cwere carried out and were followed by a cycle in which theextension reaction at 72 8C was prolonged for 15 min in orderto complete DNA synthesis. The amplified fragment waspurified by agarose gel electrophoresis, ligated into pGEM-4zvector, and sequenced. A 26-nt oligonucleotide (AAT probe,5 0-CTGTTGCTTTTGGATTACAACGCATA), chosen from thesequenced region of the PhaspC amplified fragment, was32P-5 0-end labelled and used as a specific probe for colonyhybridization in the screening of the HindIII and Pst IPs. haloplanktis TAC 125 genomic libraries. Plasmid DNA ofrecombinant colonies was then isolated and purified asdescribed by Sambrook et al. [16]. The nucleotide sequencewas determined by the dideoxynucleotide chain terminationmethod [16] with Sequenase (US Biochemical Corp.), usingM13 forward and reverse primers as well as synthetic oligo-nucleotides (PRIMM), which were designed from the previously

q FEBS 2000 Cold-adapted aspartate aminotransferase (Eur. J. Biochem. 267) 2791

determined sequence. The sequence has been deposited at theEMBL Databank with the accession number AJ001736.

Expression of PhaspC in E. coli

The vector pTrcPhaspC, used for the heterologous productionof PhAspAT in E. coli, was prepared following the procedurereported in Scheme 1. The N-terminal coding fragment ofPhaspC (250 bp long) was amplified by PCR using as templatethe plasmid derived from the PstI library screening, and thesynthetic oligonucleotides AAT probe and Bam5 0 (5 0-TAAT-GATAGGATCCACTCAATG). The latter oligonucleotide, over-lapping the putative Shine±Dalgarno sequence, was designed inorder to insert a new BamHI site (underlined in the sequenceabove) 11 bp upstream of the gene start codon. Conditions forthe PCR amplification were the same as for amplifying the gene(see above) except for the reaction volume (50 mL), the amountof DNA (100 ng plasmid), the oligonucleotide concentration(1 mm), and 20 cycles were carried out with an annealingtemperature of 50 8C. The C-terminal coding portion (H2A) ofPhaspC was obtained by HindIII digestion of the genomic

clone pH2A. The two fragments (1A/Bam and H2A) were thenligated into EcoRI/HindIII-digested pGEM-4z, thus reconstitut-ing the whole PhaspC gene. The region of the gene that hadbeen obtained by amplification was checked by direct sequenc-ing. The PhaspC coding region was then recovered frompGEM-PhaspC by EcoRI/HindII digestion and ligated into theexpression vector pTrc99A, previously digested with EcoRI andSmaI, thus generating the vector pTrcPhaspC.

Purification of recombinant PhAspAT

Recombinant PhAspAT was purified from lysates of TY103E. coli cells bearing the pTrcPhaspC plasmid. Bacteria weregrown at 37 8C in Luria±Bertani broth supplemented with100 mg´mL21 ampicillin, 30 mg´mL21 chloramphenicol,15 mg´mL21 tetracycline, and 50 mg´mL21 kanamycin.Expression was optimized by testing different incubationtemperatures and several isopropyl thio-b-d-galactopyranosideinduction conditions after inoculating an overnight culture intothe same medium at an absorbance of 0.05. The preparativeproduction of PhAspAT was carried out at 34 8C, and

Fig. 1. Nucleotide and amino-acid sequence

of PhAspAT. Nucleotides are numbered

consecutively starting in the region upstream of

the AspAT gene. Amino acids are numbered

according to the deduced start of the coding

region of the gene. The protein sequences used

to construct the oligonucleotides used as primers

in the PCR amplification are shown in bold, and

the region amplified by PCR underlined. The

oligonucleotide used in the library screening is

shaded.

2792 L. Birolo et al. (Eur. J. Biochem. 267) q FEBS 2000

expression of PhaspC was induced with isopropyl thio-b-d-galactopyranoside (1 mm) when the culture reached anabsorbance of 0.2. Cells were harvested, after incubation fora further 16 h at 34 8C, by centrifugation for 15 min, 2500 g,4 8C, and deeply frozen at 280 8C. Bacterial cells wereresuspended (3 mL per g wet cells) in 20 mm Mes (sodiumsalt), pH 6.0, containing 10 mm EDTA, 0.1 mm pyridoxamineP,5 mm 2-oxoglutarate, 1 mm dithiothreitol, and 135 mm phenyl-methanesulfonyl fluoride, and extensively sonicated keepingthe temperature constant in an ice-bath. Cell debris wasremoved by centrifugation at 11 300 g, 4 8C, for 30 min, andthe supernatant was fractionated between 30 and 80%(NH4)2SO4 saturation. Precipitated proteins were resuspendedin 20 mm Mes (sodium salt), pH 6.0, extensively dialysedagainst the same buffer, and loaded on a DEAE-Sepharose FastFlow (Pharmacia Biotech Inc.) column (2.5 � 17 cm) equili-brated in the above buffer. After the column had been washedwith 200 mL 20 mm Mes, pH 6.0, proteins were eluted with a0±0.6 m NaCl linear gradient (500 mL). Fractions containingAspAT activity were pooled, concentrated by precipitation with(NH4)2SO4 at 82% saturation followed by centrifugation for30 min, 9800 g at 4 8C. The precipitate was then resuspendedin 50 mm Tris/HCl/0.15 m NaCl, pH 7.0, and loaded on aSuperdex 200 prep grade 35/600 column equilibrated in thesame buffer. The active fractions were pooled, concentrated anddesalted by ultrafiltration on an Amicon PM-10 membrane, andloaded on an ion-exchange Poros 50 HQ column (PerSeptive

Biosystems, Framingham, MA, USA) equilibrated in Tris/HCl50 mm, pH 7.0. Proteins were eluted in a 0.15±0.25 m NaCllinear gradient (240 mL). Active fractions were pooled,desalted and concentrated by ultrafiltration on an AmiconPM-10 membrane.

Protein concentration was determined using the Bradfordprotein assay (Bio-Rad), with BSA as standard.

Purification of recombinant AspAT from E. coli

AspAT from E. coli (EcAspAT) was isolated from the over-producing E. coli strain TY103 [13] harbouring the plasmidpKDHE19 [14]. Bacteria were grown and protein purified asdescribed previously [17]. The enzyme preparations werehomogeneous on SDS/PAGE. The purified enzymes werestored at 280 8C in the presence of 1 mm dithiothreitol and anexcess of pyridoxamineP and 2-oxoglutarate.

Subunit concentrations of EcAspAT were determined spectro-photometrically on a Beckman DU7500 spectrophotometer,using :280 � 49 935 m21´cm21 [18].

Determination of molecular mass and peptide mapping

Protein molecular mass was determined by electrosprayionization (ESI)-MS using a Bio-Q triple quadrupole massspectrometer (Micromass, Manchester, UK) equipped with anelectrospray ion source. Samples were injected into the ionsource (kept at 80 8C) via a loop injection at a flow rate of

Scheme 1.

q FEBS 2000 Cold-adapted aspartate aminotransferase (Eur. J. Biochem. 267) 2793

10 mL´min21. Data were acquired and elaborated using themasslynx program, purchased from Micromass. Mass calibra-tion was performed by means of multiply charged ions from aseparate injection of horse heart myoglobin (average molecularmass 16 951.5 Da); all masses are reported as average values.

In order to check the amino-acid sequence of PhAspAT, theprotein was incubated overnight with trypsin (Sigma), using anenzyme to substrate ratio of 1 : 50. The digestion products wereanalysed by MALDI-MS on a Voyager DE MALDI TOFinstrument (PerSeptive Biosystem), equipped with a pulsednitrogen laser: 1 mL of a mixture of analyte solution anda-cyano-4-hydroxycinnamic acid (Sigma) was applied to themetallic sample plate and dried at room temperature. Massspectra were recorded using an accelerating voltage of 25 kVand a delay time of 200 ns. Mass calibration was performedusing the insulin ion at 5734.4 Da and a matrix peak at374.3 Da as internal standards. Raw data were analysed withGRAMS/386 software provided by the manufacturer and arereported as average masses.

Thermodependence of enzyme activity

To avoid any interference in the assay of possible thermo-dependence of malate dehydrogenase activity, transaminaseactivity was monitored by measuring the rate of increase inA412 when the enzyme was added to solutions containingthe less common substrate l-cysteine sulphinate (CSA) and2-oxoglutarate in 50 mm Hepes (sodium salt), pH 7.5, 0.1 mmEDTA, 0.20 mm 5,5 0-dithiobis(2-nitrobenzoic-acid). The con-centrations of CSA and 2-oxoglutarate differed depending onthe assay temperature. Units of activity were calculated usingan absorption coefficient of :412 � 14 150 m21´cm21 for the5-thio-2-nitrobenzoate formed during the reaction [19].

Apparent Km values for CSA at different temperatures weredetermined from the initial steady-state rates, varying theconcentration of CSA while keeping 2-oxoglutarate at aconstant and saturating concentration. At each temperature,the 2-oxoglutarate concentration in the assay was defined,checking that by either doubling or halving it, the reaction ratedid not vary.

Determination of maleate and a-methylaspartatedissociation constants by direct titration

A 5.7-mm PhAspAT solution in 50 mm Tes (sodium salt),pH 8.0, was titrated by directly adding small aliquots of

concentrated ligand solution, and the increase in A430 (formaleate titration) or A330 (for a-methylaspartate titration) wasmeasured. Absorbance data were fitted to the followingequation [20]:

A � A0 ÿ �A0 ÿ A1� �L�K � �L�

where A is the absorbance observed, [L] is the ligandconcentration and A0, A1 and K are adjustable parameters.

Tryptophan fluorescence and CD

In all the experiments, enzyme concentration was 3.4 mmin 10 mm Tes (pH 7.5)/1 mm dithiothreitol. Fluorescencemeasurements were carried out on a Perkin-Elmer LB50Sfluorimeter in 10 mm cells with thermostatically controlled cellholder, and temperature was measured directly in the cuvette.The solutions were filtered just before use, and data werecorrected by subtracting values for a control from which theenzyme was omitted. The bandwidths for excitation (295 nm)and emission were set to 10 and 2.5 nm, respectively, andemission spectra were collected between 310 and 480 nm ateach temperature. Ellipticity at 220 nm was measured as thetemperature was varied from 25 to 80 8C at a rate of1 8C´min21. A Jasco J715 spectropolarimeter equipped with aPeltier thermostatic cell holder (Jasco PTC-343) and a 0.1-cmpath length quartz cell were used.

Molecular modelling

A homology model for the 3D structure of PhAspAT monomerwas built using the method in the program modeler [21]incorporated in the insight II suite [22]. This approachcalculates a model structure of a target protein by satisfactionof spatial restraints drawn from a set of homologous templatestructures. The PhAspAT model was developed from fiveAspAT structures (Fig. 2): E. coli (PDB code 1ARS), chickenmitochondrial and cytosolic (7AAT, 2CST), pig cytosolic(1AJS), and yeast cytosolic (1YAA). Template sequenceswere aligned by superimposition of their Ca carbons. ThePhAspAT sequence was aligned with the superimposedtemplate sequences, and the resulting multiple alignment wasmanually refined, particularly at loop regions. Four differentPhAspAT models were calculated by modeler starting fromthe same multiple alignment, with application of the full

Fig. 2. Superimposition of the monomeric

structural templates. Traces of the five AspAT

monomers serving as templates in the homology

modelling of PhAspAT are displayed as a stereo

picture. The AspAT traces are drawn in different

colours: E. coli, green; chicken mitochondrial

and cytosolic, black and cyan, respectively;

porcine cytosolic, red; yeast cytosolic, orange.

PyridoxalP from E. coli AspAT is displayed as a

black stick-model. The E. coli trace is numbered

every 25 residues.

2794 L. Birolo et al. (Eur. J. Biochem. 267) q FEBS 2000

molecular dynamics/simulated annealing refinement [21]implemented in the program itself. The pyridoxalP moleculewas taken from the E. coli AspAT structure and included in themodel during the calculations as a `block' residue, i.e. as a rigidbody. The model displaying the lowest `objective function',indicating good satisfaction of the observed spatial restraints,was selected for further processing. Model consistency waschecked with the programs procheck [23] and prosaii [24].The PhAspAT dimer was built on to the dimeric E. coli AspATstructure. Steric clashes at the subunit interface were manuallyremoved. The dimeric model was then optimized by energyminimization: only residues at the interface were allowed tomove under a tethering forcing constant of 418 kJ´AÊ 21, and atorsional force applied to v angles with forcing constant209 kJ´rad22. A distance-dependent dielectric constant, nomorse potential, no cross-terms, and charges on were used for100 steepest-descent and 200 conjugate-gradient energy minim-ization steps. The interface was once more manually inspected,refined and then subjected to a further 10 and 20 steps ofsteepest-descent and conjugate-gradient minimizers, respec-tively, run in the same conditions. Structures of the subunitinterface before and after minimization are compared in Fig. 3.Secondary structures and solvent accessibilities were calculatedwith dssp [25] and naccess [26], respectively. All the programswere run under irix 6.2/6.5 and O2 Silicon Graphics stations.

R E S U LT S A N D D I S C U S S I O N

Purification of PhAspAT

AspAT activity was purified from an exponentially growing cellculture of Ps. haloplanktis TAC 125 by following the purifica-tion protocol described in Experimental procedures. A fairlypure protein was obtained, as assessed by SDS/PAGE (data notshown), suitable for amino-acid sequencing of the electro-blotted band, which yielded the following N-terminal sequence:MFSVLKPLPTD. The apparent molecular mass of PhAspATwas estimated to be 43 kDa by SDS/PAGE.

Cloning and DNA sequencing of PhaspC

A highly conserved region in a multiple alignment of severalmicrobial AspATs and the above N-terminal sequence werechosen to design specific primers (shown in Fig. 1) for PCRamplification of a fragment of Ps. haloplanktis TAC 125AspAT gene (PhaspC ), using PstI-digested genomic DNA astemplate.

The amplified fragment (1047 bp long) was cloned in thepGEM-4z vector and sequenced, showing high identity withthe E. coli aspC gene, coding for AspAT (55%). A 25-baseoligonucleotide was chosen in the region of Ps. haloplanktisTAC 125 amplified fragment that was less conserved withrespect to the E. coli aspC gene, and used as gene-specificprobe in the screening of HindIII and PstI genomic libraries ofPs. haloplanktis TAC 125. Four positive colonies were foundfrom 6 � 103 and 7.5 � 103 transformants screened in the twogenomic libraries, respectively. Three clones, derived from theHindIII collection, harboured the same genomic insert of about2 kb (pH2A). The positive colony isolated from the Pst I librarycarried a larger genomic insert (about 8 kb) (pP1A), encom-passing the whole PhaspC gene. Indeed, HindIII digestionshowed that pH2A is part of pP1A. A genomic region of1748 bp was sequenced, which contained the PhaspC gene(1191 bp long, coding for 397 amino acids) and its flankingregions. Figure 1 shows the PhaspC gene sequence and itstranslation.

Expression of the psychrophilic AspAT in E. coli

The PhaspC gene was cloned downstream from the Trcpromoter in the pTrc 99A expression vector (Scheme 1), andthe resulting plasmid (pTrcPhaspC ) was transferred into theE. coli host strain deficient in aspartate and tyrosine amino-transferases, TY103 [13]. Several conditions, i.e. growthtemperature, cell density and induction time, were tested tooptimize recombinant protein production. A reasonable com-promise between the likely thermolability of the psychrophilicgene product and the growth requirements of the mesophilic host

Fig. 3. Comparison of the PhAspAT model before and after energy minimization. A portion of the trace of the final dimer of PhAspAT is displayed as a

stereo picture with blue and cyan colours denoting the two subunits. Monomer before the energy minimization is drawn in red. The pyridoxalP molecule is

coloured orange. Side chains that were allowed to move during minimization (see text for details) are highlighted as stick models coloured as the monomer to

which they belong. Labels identify residues by the one-letter code and sequence position. The view is approximately perpendicular to the molecular twofold

axis of symmetry.

q FEBS 2000 Cold-adapted aspartate aminotransferase (Eur. J. Biochem. 267) 2795

was 34 8C, with a 16-h isopropyl thio-b-d-galactopyranosideinduction, starting at a cell density at A600 of 0.2 (Table 1).

The AspAT specific activity in the cell extract of E. colicells containing pTrcPhaspC was 40 times greater than the

endogenous activity in Ps. haloplanktis TAC 125 when grownat 4 8C (data not shown). Km values for l-aspartate, CSA, and2-oxoglutarate, determined at 15 8C, were identical, within therange of experimental error, for both the recombinant and theextracted PhAspAT (data not shown).

Purification and characterization of recombinant PhAspAT

Recombinant PhAspAT was purified (Table 2) from TY103E. coli cell extract by anion-exchange chromatography onDEAE-Sepharose followed by gel filtration on Superdex 200and a further anion-exchange chromatography on POROS 50HQ. A production of about 2 mg homogeneous protein per gwet cells was consistently obtained.

The molecular mass of the recombinant PhAspAT, deter-mined by ESI-MS (43 773.7 ^ 2.5), did not correspond to that

Table 1. Temperature and induction time dependence of PhAspAT

production in E. coli. Overnight cultures (37 8C) were diluted to A600 0.05

at the indicated temperature. When A600 reached the indicated value,

PhAspAT expression was induced by addition of 1 mm isopropyl thio-b-d-

galactoside, and the culture incubated for the indicated time before cell

recovery. Specific activity was measured on the cell homogenate. The

conditions chosen for large scale production are shown in bold.

Temperature

(8C)

A600 at

induction

Length of

induction (h)

Specific activity of

cell extract (U´mg21)

26 1�.30 16 2�.21

28 0�.50 16 3�.32

32 0�.55 16 8�.47

34 0.20 16 12�.92

0.45 16 10�.14

1.1 4 7�.95

1.1 6 9�.52

1.1 8 12�.14

2.0 4 5�.88

2.50 16 7�.72

4.0 7 5�.96

37 0�.42 16 7�.89

2.70 16 5�.88

Table 2. Purification of recombinant PhAspAT.

Purification step

Total

activity

(U)

Total

protein

(mg)

Specific

activity

(U´mg21)

Recovery

(%)

Cell lysate

(NH4)2SO4 35%

1357

1249

331�.7

277.9

4�.1

4.5

100

92

DEAE-Sepharose

(NH4)2SO4 82%

1259

1011

57�.1

44.7

22�.1

22.6

93

74.5

Superdex 200 955 15�.5 61�.7 70�.4

POROS HQ 690 5�.0 137�.7 50�.8

Fig. 4. Amino-acid alignment of the psychrophilic PhAspAT with the mesophilic EcAspAT. The 11 amino-acid residues that are absolutely invariant in

all the AspATs sequenced so far [27] are shown in bold. The secondary-structure elements derived from the structure of EcAspAT [11] are reported. Symbols

are h, e, t, and blank for a-helix, b-strand, b-turn, and random coil, respectively.

2796 L. Birolo et al. (Eur. J. Biochem. 267) q FEBS 2000

calculated from the nucleotide sequence. Complete massmapping of the recombinant protein (data not shown) led tothe definition of two ambiguities in the nucleotide sequence.The revised sequence is shown in Fig. 1, where the correctedcodons are boxed. The expected average molecular mass(43 776.14) is in good agreement with the experimental value.

PhAspAT sequence alignment

The amino-acid sequence of PhAspAT was aligned with that ofseveral other AspATs. PhAspAT shows the highest identitywith the mesophilic E. coli enzyme (52%). The alignment ofPhAspAT with the E. coli enzyme is shown in Fig. 4, in whichthe 11 invariant residues in all AspATs sequenced so far arehighlighted in bold [27]. The residue numbering refers thealignment of EcAspAT to porcine cytosolic AspAT [28].

A total of 38 AspAT sequences from various organisms wereretrieved from the SwissProt Data Bank and aligned to thePhAspAT sequence with the program clustalw (http://www2.ebi.ac.uk/clustalw/). Conserved amino acids are evenlydistributed along the enzyme sequence. Sequence identitybetween PhAspAT and the other AspATs ranges from 12.0%for AspAT2 from Methanococcus jannaschii to 52.0% forEcAspAT. From these sequences, two groups can be clearlydistinguished: 10 AspATs share less than 15.0% identity withPhAspAT, whereas the others display more than 32.0% identity.AspATs from Bacillus subtilis, Methanococcus jannaschii,Bacillus spp., Bacillus stearothermophilus, S. solfataricus,Thermus thermophilus, and Rhizobium meliloti belong to themost distant group, essentially arising from thermophilicorganisms.

The average amino-acid composition of the 38 homologousAspATs, except PhAspAT, was determined, and the deviationof PhAspAT from the average composition was calculated andexpressed in units of standard deviations for each of the 20amino-acid residues, where positive or negative values repre-sent higher or lower occurrence than average, respectively. Theanalysis shows that Cys and Leu tend to be more represented(standard score 11.9 and 11.6, respectively) while Pro, Phe,Gly are less common, although with different statisticalsignificance (respective standard score: 21.7, 21.2, 21.0).However, care should be taken in interpreting these data asrelated to cold adaptation because residue compositionalanalysis may not provide sound information on the molecularmechanisms of adaptation to extreme environments [29]. Onlythe comparison of 3D structures can rationalize the observedstatistics based on sequence alignment analysis.

Structural model analysis

The favourable degree of identity with EcAspAT allowed thebuilding of a model of the 3D structure of PhAspAT. The finalmodel displays good stereochemical quality, with a prosaii [24]energy profile comparable with that of the EcAspAT structure.The predicted molecular architecture follows the pattern ofknown AspAT structure, with conservation of the main featuresof the active-site region. The interactions between pyridoxalP,substrate and enzyme active-site residues are therefore fullyconserved. Notably no extra or longer loops, structural elementsthat could conceivably contribute to protein flexibility, havebeen found.

Structural analysis of the model indicates a few sites wherethe physicochemical nature of the corresponding side chain inPhAspAT is different from that conserved in all, or most, otherAspATs (Table 3). In this respect, the interface region between

the two subunits in the dimeric form of the enzyme seems to beparticularly affected. Residues at the subunit interface, definedas those amino acids that lose surface accessibility after subunitassociation, are reported in Table 4. Some of the observedreplacements produce a decrease in the volume of the sidechain, possibly introducing cavities and looser packing of thesubunits. These replacements are briefly reported and com-mented on below. Cysteine replaces the normally occurringbulky and/or hydrophobic residues in position 122 (accordingto residue numbering of porcine cytosolic AspAT [28]). Aserine replaces the highly conserved threonine in position 196,at a site at the interface between the large and the small domain,which undergoes a structural rearrangement on substratebinding [9]. A leucine replaces the conserved Pro200, whichmay affect chain flexibility. An alanine occurs in position 212,in a buried a-helical site where large hydrophobic residues areusually found in the group of more related AspATs. Likewise, athreonine replaces hydrophobic residues at position 218. Thismutation, at the interface to the N-terminal arm of the othersubunit, may greatly decrease the interaction with Phe6 fromthe adjacent subunit which is conserved in most of the relatedAspATs. A valine replaces a Phe at position 221, interacting

Table 3. Potentially important residue substitutions in PhAspAT.

Residue Structural environment

Corresponding observed

residues in the most

closely related AspATs

60 Arg a-Helix; subunit interface Ile, Met, Leu

122 Cys C-Terminal of a-helix;

subunit interface at

N-terminal arm

Phe, Tyr, Trp, His, Val, Leu,

Ile, Gln, Asn

196 Ser Turn; interface

large/small domain

Thr

200 Leu Buried site in a loop Pro

212 Ala a-Helix; partly buried Ser, Met, Val, Ile, Phe

218 Thr Loop; interface to the

N-terminal arm

Phe, Val, Ile, Leu

221 Val b-Strand; buried Val, Phe

256 Cys a-Helix; buried Phe, Tyr

264 Arg Turn; subunit interface Gly, Ala, Asn

Table 4. Subunit interface residues, pyridoxalP form of PhAspAT

model.

Residues associated with the N-terminal arm:

Met5, Phe6, Ser7, Leu9, Lys10, Pro11, Leu12, Phe118, Ile119a, Arg121,

Cys122, Phe217a, Thr218, Glu249, Leu250a, Ile251a, Ile273a, Ala274,

Lys275, Thr279, Ile282, Ser283a, Ser285, Val286, Ser289

Residues forming the interface around the dimer twofold axis:

Asp15, Ile17a, Leu18, Met21, Val37a, Gly38, Val39, Lys41a, Asn46,

Thr47, Pro48a, Val49, Leu50a, Val53a, Lys54, Glu57, Ala58, Arg60,

Leu61, Glu64, Thr66, Ser67, Lys68, Ser69, Tyr70, Ile71, Leu73,

Pro106, Thr109, Gly110a, Arg113, Glu117, Trp140, Ala141a,

Asn142, Ser145, Leu146, Glu148a, Ala149, Ala150a, Ser257a,

Lys258a, Gly261, Leu262a, Tyr263, Arg264, Glu265, Arg266,

Leu272, Arg292, Ser293, Ile294, Tyr295, Ser296, Met297, Pro298a,

Pro299, Ala300, His301, Asp304

a Residues losing less than 10 AÊ 2 on dimer formation.

q FEBS 2000 Cold-adapted aspartate aminotransferase (Eur. J. Biochem. 267) 2797

with the highly conserved Trp205. A cysteine is found atposition 256, replacing Phe, Tyr, Val, or Ala, and disrupts thehydrophobic interaction with Phe, Leu, Tyr, or Met which arefound differently at position 79.

Other substitutions introduce a charged amino acid inpositions where neutral or polar residues are generally located.Two arginine residues are found at positions 60 and 264 withina non-polar environment and their positively charged sidechains are probably neutralized by salt bridges, possibly withGlu64 (from the partner subunit) and Asp304, respectively.

Comparison of the accessible surface area lost by monomerson dimerization in the five template structures shows thatS. solfataricus AspAT [12] and PhAspAT lose 19.63% and17.5% of the area, respectively. EcAspAT monomer losesonly 16.7% of total area, while chicken cytosolic AspATdisplays the highest loss with 20.0%. The contribution ofaromatic residues (Phe, Trp, Tyr) to the subunit interface hasbeen analysed. The lowest percentage is observed forPhAspAT, where only 12.4% of the interface surface consistsof aromatic residues whereas the highest contribution can be

Fig. 5. Interface subunit. Stereo picture of

(A) PhAspAT dimer interface compared with

the equivalent portions from (B) EcAspAT and

(C) AspAT from Sulfolobus solfataricus. Thick

and thin lines distinguish the two dimer

subunits. Residues are labelled using the

one-letter code and those from the other subunit

are marked `B'. Figures were drawn with

molscript [37].

2798 L. Birolo et al. (Eur. J. Biochem. 267) q FEBS 2000

observed in the case of S. solfataricus AspAT (24.5%), as wehave already described [12]. In contrast, basic residues (Arg,Lys, His) give the lowest contribution to the S. solfataricusdimer interface surface (14.4%) and the highest to PhAspAT(25.7%). A comparison of the subunit interfaces is shown inFig. 5.

Effect of temperature on enzyme activity

The apparent Km values of PhAspAT and EcAspAT for CSA, atsaturating 2-oxoglutarate concentrations, were determined atdifferent temperatures in the 7±45 8C range (Table 5) using theenzyme assay described in [30]. In the above range, the Km forCSA largely increases with temperature for the psychrophilicenzyme, whereas it is almost constant for EcAspAT. This kindof temperature dependence of the substrate affinity has alsobeen reported for other enzymes from psychrophilic organism[4]. It is worth mentioning that the saturating concentration of2-oxoglutarate increases with temperature (see Experimentalprocedures), suggesting a decrease in affinity also towards theoxo substrate.

Figure 6 shows the thermodependence of the initial trans-aminating velocity (kcat) towards CSA in the range 5±70 8C.The apparent maximum catalytic activity was observed at 64and 75 8C for PhAspAT and EcAspAT, respectively. The quitehigh temperature of the maximum of kcat is in agreement withthe general tendency to thermophilicity of AspATs that usuallyexhibit maximum activity at about 30±40 8C above theoptimum growth temperature of the organism [31]. However,because of the large temperature effect on the Km, themaximum catalytic efficiency (inset Fig. 6) occurs at 30 8C.

It is worth pointing out that both kcat and kcat /Km values arelower than those of EcAspAT over the whole temperature range.Thus, higher catalytic efficiency, usually claimed as a charac-teristic of psychrophilic extracellular enzymes, evidently doesnot apply to PhAspAT (see Conclusions).

Effect of temperature on substrate affinity

The half-reaction pathway of enzymatic transamination isreported in Scheme 2, showing the various intermediates in theconversion of aspartate to oxaloacetate. In order to investigatethe temperature effect on the affinity of PhAspAT forsubstrates, we isolated some of the above intermediates usingsubstrate analogues.

Maleate, a dicarboxylate substrate analogue, is known tobind abortively to the pyridoxal form of the enzyme (internalaldimine), inducing the open to closed conformational changeof the enzyme and increasing the pKa of the imine [32,33].Figure 7 shows the dissociation constant (Kd) of the PhAspAT±maleate complex measured at different temperatures, demon-strating invariance of Kd with temperature in the range tested.

The amino acid 2-methyl-l-aspartate binds to the enzymeactive site to form a Michaelis complex, which undergoestransaldimination to the external aldimine, which is the firstobligatory step in transamination (Scheme 2). The subsequentstep to form the semiquinonoid intermediate is impairedbecause of the absence of the hydrogen on the a-carbon.Again, the Kd of the PhAspAT22-methyl-l-aspartate complexat different temperatures (Fig. 7) showed no change withtemperature.

These results show that, at least in the temperature rangetested, formation of both the Michaelis complex and theexternal aldimine is not affected by temperature, opposing thenaive hypothesis that progressive loosening of the enzymearchitecture makes the fit of the substrate to the active site lessand less tight. The observed increase in Km can be explained bya temperature effect at any of the steps in the catalytic pathwayafter the transaldimination to the external aldimine, rather thanby the actual binding substrate. Further investigations areneeded to elucidate more precisely this interesting phenomenonand to understand the physiological significance, if any,behind it.

Thermal stability of PhAspAT

Tryptophan fluorescence and amide ellipticity were used tofollow changes in tertiary and secondary structure of PhAspATas a function of temperature, respectively. PhAspAT has fourtryptophan residues, which, on the basis of the homology model(see above), are evenly distributed among different regions ofthe 3D structure, with one of them located in the active-sitepocket. Therefore, tryptophan fluorescence is considered here,to a first approximation, to be an excellent probe for monitoringglobal changes in tertiary structure during protein unfolding.Fluorescence intensity at 333 nm (wavelength corresponding

Fig. 6. Temperature dependence of transamination. kcat and kcat/Km

(inset) for the transamination of the substrate CSA for PhAspAT (s) and

EcAspAT (X) were determined at increasing temperature as described in

Experimental procedures.

Table 5. Thermodependence of substrate affinity of PhAspAT and

EcAspAT. ND, not determined.

Temperature

Km (CSA) (mm)

(8C) PhAspAT EcAspAT

7 5.82 �^ 0.24 ND

15 3.35 �^ 0.37 ND

25 8.34 �^ 0.52 7.31 �^ 0.28

35 21.04 �^ 1.4 7.31 �^ 0.23

45 64.7 �^ 6.7 6.54 �^ 0.46

q FEBS 2000 Cold-adapted aspartate aminotransferase (Eur. J. Biochem. 267) 2799

to the emission maximum at 20 8C of both PhAspAT andEcAspAT) rapidly increases with temperature in the range45±55 8C and 55±70 8C for PhAspAT and EcAspAT, respec-tively (Fig. 8A), with a concomitant shift in maximum emissionwavelength from 333 to 344 nm (Fig. 8A). A transitiontemperature at about 50 8C and 65 8C can be observed in thefluorescence melting curves for PhAspAT and EcAspAT,respectively.

Figure 8B shows the changes in secondary structure forPhAspAT and EcAspAT as a function of temperature. Themelting temperature of 67 8C calculated for EcAspAT is ingood agreement with the value (65.5 8C) determined by

differential scanning calorimetry [34]. These authors alsoclearly showed that only the Tm (midpoint of thermal transition)value could be taken into consideration, as they observed the

Scheme 2.

Fig. 7. Temperature dependence of substrate binding. Km values for

PsAspAT for CSA (X), and Kd values for maleate (B) and a-methyl-

aspartate (m) were determined at increasing temperature as described in

Experimental procedures.

Fig. 8. Thermal transition curve of AspATs from Ps. haloplanktis

TAC 125 and E. coli. Enzyme concentration was 3.4 mm in 10 mm Tes

(pH 7.5)/1 mm dithiothreitol. (A) Fluorescence intensity of samples of

PhAspAT (s) and EcAspAT (X) incubated at different temperatures was

recorded at 333 nm (excitation 295 nm). The wavelength of the emission

maximum is also reported for PhAspAT (B). (B) CD at 220 nm of

PhAspAT (solid line) and EcAspAT (broken line) was recorded as

temperature was increased at a rate of 1 8C´min21.

2800 L. Birolo et al. (Eur. J. Biochem. 267) q FEBS 2000

occurrence of at least two endotherms and the lack of fullreversibility. The CD-detected unfolding curve of PhAspAT iseven more complex than that of EcAspAT, as shown in Fig. 8B.One can clearly see the occurrence of at least two differentprocesses, well separated on the temperature scale. A firsttransition occurs at about 52 8C, some 15 8C below theEcAspAT melting point, while a second transition occurs at20 8C above.

Therefore, both tryptophan fluorescence and amide ellipticitysuggest that a structural transition occurs at about 50 8C forPhAspAT, with a concomitant loss of tertiary and somesecondary structure. However, PhAspAT retains a certainamount of secondary structure at temperatures even higherthan that of EcAspAT melting.

Thermoresistance of PhAspAT was tested by incubating theenzyme at 50 8C and assaying the residual enzymatic activityafter different incubation times. A half-life of inactivation of6.8 min at 50 8C was calculated for PhAspAT, whereasEcAspAT can be incubated under the same conditions formore than 3 h without any detectable change in activity (datanot shown).

It is also worth remembering that the inactivation tempera-tures for PhAspAT and EcAspAT are 48 8C and 62 8C,respectively [31]. These results are in agreement with theexpected thermolability of a psychrophilic enzyme.

C O N C L U S I O N S

Rapid inactivation at moderately high temperature and highspecific activity at low temperature are generally reported as themain features of cold-adapted extracellular enzymes [4].

PhAspAT is the most thermolabile, but not the most efficient,of the AspATs so far investigated with respect to structure/function/stability. Our results clearly suggest that the overallPhAspAT architecture is more sensitive to temperature than thatof EcAspAT, the most extensively investigated bacterialtransaminase.

Molecular modelling suggests the occurrence of somecavities and loosening of side chain packing in some cruciallocations. This may eventually be considered to be analternative strategy to adequately adapt catalysis to the lowenvironmental temperatures. However, this kind of structureloosening does not apparently stem from the need for extremelyhigh efficiency. The optimum temperature for activity isactually shifted downward with respect to the E. coli enzymeboth when the catalytic constant (kcat) and the catalyticefficiency (kcat /Km) are considered, but both kcat and kcat /Km

are significantly lower than those of EcAspAT over the wholetemperature range examined. In other words, in its adaptation tocold, AspAT seems to have evolved towards a more thermo-labile protein rather than towards a more efficient biocatalyst.In this respect, PhAspAT seems to behave in the opposite wayto thermophilic enzymes, which need to evolve primarilytowards a thermostable structure while shifting upward theiroptimal temperature. Cold-loving enzymes, particularly thesecreted ones, have to cope with reduced reaction rates at lowtemperatures and thus they have to improve their catalyticefficiency. However, although thermal instability has beenobserved for all the enzymes so far characterized frompsychrophilic organisms, high specific activity at low tempera-ture is not evident either for PhAspAT or a psychrophilic triosephosphate isomerase [35] or a psychrophilic citrate synthase[36]. The fact that these three enzymes are all cytosolic is worthconsidering. One can speculate that the thermolability ofendocellular cold-adapted enzymes could be an effect of the

lack of selective pressure towards thermal stability [3] ratherthan the consequence of the structural relaxation required toperform an efficient catalytic act at low temperatures. In fact,although maximum efficiency is required for extracellularcatalysts, mainly because of their nutritional importance,endocellular ones may not have been subjected to such severeenvironmental pressure.

A C K N O W L E D G E M E N T S

The authors acknowledge the assistance of Alessia Errico and Andrea

Scaloni in protein sequencing and peptide mapping, Francesco Serci for

molecular modelling, and Giulio Gianese for use of his unpublished

programs for residue compositional analysis. This work was supported by

the European Union contracts ERBBIO4CT950017 `EUROCOLD' Con-

certed Action, ERBBIO4CT960051 `COLDZYME', ERBFMRXCT970131

`COLDNET', CNR contract 97.01138.PF49 `̀ BIOTECNOLOGIE',

MURST PRIN 1999.

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