Eur. J. Biochem. 220, 543-549 (1994) 0 FEBS 1994

Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus Zitoralis Giuseppina ANDREOTTI', Maria Vittoria CUBELLIS ', Gianpaolo NITTI*, Giovanni SANNIA', Xuhong MAI', Gennaro MARINO' and Michael W. W. ADAMS' ' Dipartimento di Chimica Organica e Biologica, Universitii di Napoli, Italy

CEINGE-Biotecnologie Avanzate, Napoli, Italy Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens GA, USA

(Received October 11December 8, 1993) - EJB 93 1535/4

The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98°C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of K,,, and kc,, values using 2-oxoglutarate, phenylalanine, tyro- sine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcar/K,,, =400 s-' mIv-') ; the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one. Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30°C and exhibited maximal activity between 95- 100°C. They showed no N-terminal sequence similarity with each other (-30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escher- ichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic 7: litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indole- pyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.

Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of grow- ing at temperatures near and even above 100 "C [ l -41. Virtu- ally all of them are classified as Archaea (formerly Archae- bacteria [5]). They include both methanogenic and sulfate- reducing genera, but the majority are sulfur-dependent organ- isms that reduce elemental sulfur ( S O ) to H,S. Most of the So-dependent species are strictly anaerobic heterotrophs that utilize peptides and proteins as their carbon and nitrogen

Correspondence to M. W. W. Adams, Department of Biochemis- try, Life Sciences Bldg., University of Georgia, Athens, GA 30602, USA

Fax: f l 706 542 0229. Abbreviations. ArAT, aromatic aminotransferase ; ArATEc, aro-

matic aminotransferase from Escherichia coli; ArAT-Ma, aromatic aminotransferase I from Methanococcus aeolicus; ArAT-IIMa, aro- matic aminotransferase I1 from Methanococcus aeolicus; AiAT-127, aromatic aminotransferase I from Thermococcus litoralis ; ArAT- IITZ, aromatic aminotransferase I1 from Thermococcus litoralis ; AspAT, aspartate aminotransferase ; AspATEc, aspartate aminotrans- ferase from Escherichia coli ; TyrAT, tyrosine aminotransferase ; TyrATEc, tyrosine aminotransferase from Escherichia coli.

Enzymes. Aromatic aminotransferase (EC 2.6.1.57) ; aspartate aminotransferase (EC 2.6.1 .l) ; tyrosine aminotransferase (EC 2.6.1.5).

sources [2-41. Some are also able to utilize certain carbohy- drates as a carbon source and a novel pathway for carbohy- drate metabolism has been proposed [6-8). The pathways of peptide utilization are less understood. Several hyperther- mophilic archaea have been shown to contain high intracellu- lar proteolytic activities [4, 9, 101 together with high concen- trations of glutamate dehydrogenase [4, 11 -141. In addition, a novel tungsten-containing enzyme has been proposed to play a role in peptide metabolism [15]. However, the mecha- nisms by which amino acids are metabolized are not known. We report here on two aminotransferase-type enzymes that transform aromatic amino acids in the hyperthermophilic archaeon Thermococcus litoralis, a marine organism that grows up to 98°C by the fermentation of peptides [16].

Aminotransferases are pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible transfer of the amino group from an amino donor to an amino acceptor [17]. They are widely distributed in both prokaryotic and eukaryotic or- ganisms, and are directly or indirectly involved in the biosyn- thesis of most amino acids and take part in the catabolism of some of them. The activity of aminotransferases that utilize aromatic amino acids has been measured in several meso- philic microorganisms and some of them have been purified [17 -211. However, their characterization has been compli-

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cated by the fact that two or more enzymes with overlapping specificities are often found in the same microorganism. For example, in Escherichia coli there are two aminotransferases that utilize both aromatic amino acids and aspartate, one encoded by the gene tyrB, the other encoded by the gene aspC [22]. The former enzyme is referred to as TyrATEc or ArATEc and is more active on tyrosine, while the second enzyme or AspATEc is more active on aspartate [17, 231. Biosynthesis of the two enzymes is regulated independently as the expression of TyrATEc is repressed by tyrosine while that of AspATEc is constitutive [22, 231.

On the other hand, four distinct aminotransferases have been detected in the methanogenic archaeon Methanococcus aeolicus, and these utilize branched-chain amino acids, aspartate and aromatic amino acids [20]. Two of them prefer- ably utilize aromatic amino acids and are referred to as ArAT- Ma and ArAT-IIMa. The other two enzymes preferentially catalyze the transamination of either branched-chain amino acids or aspartic acid, but show much lower activity towards aromatic amino acids. In all cases, the prokaryotic ArATs characterized so far exhibit significant transaminase activity with all three of the aromatic amino acids. In contrast, mam- malian systems contain an aminotransferase specific for tyrosine as the substrate termed tyrosine aminotransferase (TyrAT) [24]. This enzyme is hormonally regulated and the gene for the rat liver enzyme has been cloned, sequenced and expressed in E. coli (see [25]).

It was therefore of some interest to investigate the nature of the aromatic aminotransferases in a hyperthermophilic archaeon such as 7: litoralis, both for comparison with ArATs from prokaryotic and eucaryotic sources, and to provide in- formation on the pathways of aromatic amino acid metabo- lism in this unique group of proteolytic organisms.

MATERIALS AND METHODS Materials

Q-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, Phenyl-Sepharose Fast Flow, Superdex-200 HiLoad (1 6/60) and Ampholine were purchased from LKB-Pharmacia; phen- azine methosulfate, p-iodo-nitrotetrazolium violet, L-amino acids, 2-oxoglutarate and pyridoxamine 5'-phosphate from Sigma; AG 1 -X8 200-400 mesh formate form was obtained from Bio-Rad, and 2-0xo['~C]glutarate (56.5 Ci/mol) from Dupont NEN Research Products.

Purification procedure Thermococcus litoralis (DSM 5473) was grown as de-

scribed previously [15]. Wet cells (20 g) were resuspended in 60 ml SO mM Tris/HCl pH 8.0 containing 2 mM sodium dithionite and homogenized with lysozyme (0.5 mg/ml) and DNase (0.5 pglml) at 37°C for 1.5 h. A cell-free extract was obtained by centrifugation at 19000 rpm for 25 min and the supernatant was then dialyzed against 50 mM Tris/HCl pH 8.0. All the purification steps were performed at 23°C. Pyridoxamine 5'-phosphate (pyridoxamine-P) and 2-oxoglu- tarate were added to all the active fractions after each purifi- cation step (to final concentrations of 0.1 mM and 2.0 mM respectively). Gradient elution separations on conventional and FPLC columns were controlled using a Pharmacia-LKB FPLC system.

Q-Sepharose Fast Flow chromatography. The dialyzed cell-free extract was loaded (2 ml/min) onto a column of

Q-Sepharose Fast Flow (2.4X 16 cm) equilibrated with 50 mM TriskICl pH 8.0 (buffer A). The column was washed with the equilibration buffer until the absorbance at 280 nm returned to baseline, and retained proteins were eluted with a linear gradient of 0.1 -0.5 M NaCl (300 ml, 2 mumin) in buffer A. Two active peaks eluted at two different ionic strengths were obtained. These will be referred to as ArAT- IT1 (peak I) and ArAT-IITE (peak 11). The active fractions from each of the two peaks were pooled separately and each was diluted 1 : 3.5 with buffer A. ArAT-IT1 and ArAT-IITI were each purified separately using the following procedure.

DEAE-Sepharose Fa y t Flow chromatography. The pooled samples of ArAT-I or ArAT-I1 from the Q-Sepharose Fast Flow step were each loaded onto a column of DEAE- Sepharose Fast Flow (2.4X 11 cm) equilibrated with buffer A, at a flow rate of 3 ml/min. The column was washed with 1 bed volume of buffer A, and the absorbed proteins were eluted with a linear gradient from 0.1 -0.5 M NaCl (450 ml, 3 ml/min) in buffer A.

Phenyl-Sepharose F a t Flow chromatography. Active fractions from the previous column were pooled and solid (NH,),SO, was added to reach a final concentration of 1.3 M. After stirring for 3 h at 4"C, the sample was filtered using 0.2-pm disposable filters and then loaded at a flow rate of 1 ml/min onto a column of Phenyl-Sepharose Fast Flow (1.4X 10 cm) which had been equilibrated with buffer A con- taining 1.3 M (NH,),SO,. The column was then washed with one bed volume of the equilibration buffer and ArAT activity was eluted with a 250-ml gradient of 1.2-0 M (NH,),SO, in buffer A at 1 ml/min. The active fractions were pooled, extensively dialyzed against buffer A containing 0.1 mM pyridoxamine-P and 2 mM 2-oxoglutarate, and then concen- trated by ultrafiltration on a Diaflo PM-1 0 membrane.

Superdex-200 chromatography. The concentrated sam- ples corresponding to ArAT-IT1 and ArAT-IITI were each loaded in several steps (1.5 mg total protein for each step) onto a Superdex-200 HiLoad (1 6/60) column equilibrated with buffer A containing 0.2 M NaCl. The flow rate of the eluent was 1 ml/min. The active fractions for the two en- zymes were each pooled separately and used directly for the next step.

Mono-Q chromatography. The sample corresponding to ArAT-IT1 was diluted 1 : 3 with 50 mM potassium phosphate pH 7.0 and loaded onto a Mono-Q column (HR 5/5) equili- brated with the same buffer. The column was washed with the equilibration buffer and the enzyme eluted with a 20-ml linear gradient of 0.1-0.15 M NaCl in the same buffer at 1 ml/min.

The sample corresponding to ArAT-IITI was dialyzed against 50 mM potassium phosphate pH 6.5 and then loaded onto a Mono-Q column (HR 5/5) equilibrated with the same buffer. After washing with several bed volumes of the load- ing buffer, the enzyme was eluted with a 40-ml linear gradi- ent of 0-0.5M NaCl in the same buffer at 1 ml/min. The active fractions of both ArAT-IT1 and ArAT-IITI were then pooled separately, dialyzed against 50 mM Tris/HCI pH 8.0, concentrated by ultrafiltration and stored at -20°C.

Enzymatic assay Enzymatic activity towards phenylalanine, tyrosine and

tryptophan was determined using a modification of the assay described by George et al. [26]. This is based on the arsenate- catalyzed formation of aromatic 2-oxoacid-end-borate com- plexes that show characteristic absorption at 330, 310 and

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300 nm for indolepyruvate, p-hydroxyphenylpyruvate and phenylpyruvate complexes, respectively. The reaction mix- ture (0.8 ml) containing 4.7 mM 2-oxoglutarate, 62.5 pM pyridoxamine-P and either 66 mM tryptophan, 12 mM tyro- sine or 49 mM phenylalanine, in 200 mM potassium phos- phate pH 7.6. The concentrations of the aromatic amino acid substrates used were a compromise between their solubilities and their determined K,,, values as described below. The reac- tion was initiated by addition of the enzyme. After incubation at 78°C for 5 min, the reaction was stopped with the addition of 0.2 ml 20% (mass/vol.) trichloroacetic acid and the mix- ture was rapidly cooled in ice. After centrifugation, 0.1 ml of the supernatant was mixed with 0.5 mi of a solution of 1 .0 M sodium arsenate, 1 .0 M boric acid, pH 6.5. The mixture was allowed to stand at 23°C for 25 min and the increase in ab- sorbance was measured. One unit is defined as the amount of enzyme that catalyzes the transamination of 1 pmol sub- strate/min under these conditions.

To determine the kinetic pararnetcrs and to investigate the reaction mechanism, the concentrations of 2-oxoglutarate and amino acids were varied in the routine assay a5 described below. In order to determine the activity towards different amino acid substrates such as alanine, aspartate, isoleucine, and valine using 2-oxoglutarate as an amino acceptor, the transamination of 2-0xol'~C]glutarate (4.7 mM, 56.5 Ci/mol) was measured [17]. This was carried out at 78°C in the pres- ence of the amino acid (120mM) and pyridoxamine-P (62.5 pM) using potassium phosphate pH 7.6 in a final vol- ume of 0.2 ml. The reaction was stopped by addition of 0.8 ml 1 M HCOOH and the unreacted 0x0 acid was sepa- rated from ['4C]gluta~nate on an anion-exchange resin (AG 1-XS) equilibrated with 1 M HCOOH containing 10 inM glu- tamate [17]. The radioactivity of the amino acid product eluted with 1 M HCOOH was measured using a Beckman LS-1701 scintillation counter.

Isoelectric point determination

Isoelectric points were determined using a linear pH gra- dient in the range 4.0-5.5 on a polyacrylamide gel slab. Electrophoresis was carried out using a Multiphor apparatus from LKB, following the manufacturer's instructions. After focusing, the pH along the gel surface was measured using a surface electrode. The enzymes were located by incubating the gel at 50°C in 20 mM 2-oxoglutarate, 4 pM pyridox- amine-P, 50 mM potassium phosphate pH 7.6, 20 pg/ml phenazine methosulfate, 200 pg/ml p-iodo-nitrotetrazolium violet and using either 10 mM 3-iodo-tyrosine or 25 mM phenylalanine or 18 mM tryptophan. In separate experi- ments, the proteins were detected by using silver staining.

Estimation of molecular mass

Molecular masses were estimated by gel filtration on a Superdex-200 HiLoad (1 6/60) column which had previously been calibrated with carbonic anhydrase (29 kDa), oval- bumin (45 kDa), bovine serum albumin (66 kDa), porcine cytosolic aspartate aminotransferase (92 kDa) and alcohol dehydrogenase from yeast (1 50 kDa). Blue dextran and pyri- doxamine-P were used to determine the void volume and the total volume, respectively. The flow rate of the eluent, 50 mM TridHC1 pH 8.0 containing 0.2 M NaCl, was 1 ml/ min. The molecular mass was calculated under denaturing conditions by SDSPAGE from slab gels containing 12.5%

Fraction number

Fig. 1. Resolution of two aromatic aminotransferases from Z litoralis on Q-Sepharose Fast Flow. Fractions (2 nil) were collected and assayed for aminotransferase activity towards phenylalanine (-), tyrosine (----) and tryptophan (. . . . . .) using 2-oxoglu- tarate as a co-substrate at 78°C under standard assay conditions. Also shown is the protein elution profile (-, A,,,,) and the salt gradient that was used (0-0.1 and 0.1 -0.5 M NaCl).

acrylamide as described by Weber et al. [27]. Proteins were located using the silver staining technique [28].

Other methods Ultraviolethisible absorption spectra of purified ArAT-

IITl (1 mg/ml in 50 mM Tris/HCI pH 8.5) were obtained on a Beckman diode array spectrophotometer (model DU 7500). Purified proteins were electroblotted from the SDS/poly- acrylamide gel onto a ProBlott membrane (Applied Biosys- tem). Transfer was conducted as described by Matsudaria [29]. N-terminal sequences were determined by automated Edman degradation using an Applied Biosystem sequencer fitted with an on-line phenylthiohydantoin amino acid ana- lyzer. Protein concentration was determined using the Bio- Rad protein assay system [30] with bovine serum albumin as the standard.

RESULTS AND DISCUSSION Purification of ArAT-IT1 and ArAT-IITZ

Two aromatic aminotransferase activities were identified when cell-free extracts of 7: litoralis were subjected to an- ionic-exchange chromatography on Q-Sepharose Fast Flow (Fig. 1). These were collected separately and designated as ArAT-IT1 and ArAT-IITI. Once separated, the relative phenyl- alanine-, tyrosine- and tryptophan-dependent aminotransfer- ase activities associated with both ArAT-IT1 and ArAT-IITI remained constant throughout the purification process and were the same as those of purified enzymes (see below). These results suggest that two distinct aromatic aminotrans- ferases exist in 7: litoralis.

The results of typical purifications for ArAT-IT1 and ArAT-1177 based on phenylalanine aminotransferase activity are shown in Tables 1 and 2. These data indicate that most and probably all of this activity in the cell-free extract is accounted for by ArAT-IT1 and ArAT-IIT1, which represent approximately 58 % and 42%, respectively. Overall, purified ArAT-IT1 represented more protein than ArAT-IITI but the latter had a much higher spedific activity than the former.

546

Table 1. Purification of ArAT-I from T. litoralis.

Purification Volume Activity Total Protein Total Specific Recovery Purifi- step activity protein activity cation

nil Ulml U mglml n3g U/mg % -fold - - Cell-free extract 80 25.3 2024 12.13 970.4 2.1

Q-Sepharose 60 14.2 850 2.21 132.6 6.4 100 1 DEAE-Sepharose 60 11.6 697 1.54 92.1 7.5 82 1.2 Phenyl-Sepharose 10 25.8 258 1.51 15.1 17.1 30 2.7 Superdex-200 0.86 144.0 124 3.2 2.8 45.0 15 7.0 Mono-Q 2.34 17.4 41 0.16 0.4 110 5 17.2

Table 2. Purification of ArAT-I1 from T. litoralis.

Purification Volume Activity Total Protein Total Specific Recovery Purifi- step activity protein activity cation

ml U/ml U mg/ml mg U/mg % -fold - - Cell-free extract 80 25.3 2024 12.13 970.4 2.1

Q-Sepharose 58 10.7 61 9 1.72 99.8 6.2 100 1 DE AE-Sepharose 57 9.2 526 0.95 54.2 9.7 85 1.6 Phenyl-Sepharose 2.8 90.0 252 1.21 3.4 74.4 41 12.0 Superdex-200 1.1 215.4 237 0.90 0.99 239.3 38 38.6 Mono-Q 0.67 79.1 53 0.19 0.13 424.0 9 68.4

A B

t 29

Fig. 2. SDSE'AGE analysis of purified aromatic aminotransfer- ases from T. litoralis. Samples from the Mono-Q chromatography were subjected to SDSPAGE on a 12.5% polyacrylamide gel. Lane A, ArAT-IT/; lane B, ArAT-IITI. The values shown correspond to the molecular masses of protein standards in kDa.

Using the procedures outlined in Tables 1 and 2, ArAT-IT1 and ArAT-IITl were each obtained in an homogeneous form as determined by SDS/PAGE electrophoresis.

Structural properties

The molecular inasses of ArAT-IT1 and ArAT-IITI were 11 0 2 5 kDa and 92 % 5 kDa, respectively, as determined by Superdex-200 gel filtration, and 47 % 3 kDa and 45 ? 1 kDa, respectively, as determined by SDSPAGE (Fig. 2). Edman degradation analysis of each enzyme produced a single un- ambiguous and distinct sequence and there was no similarity between them. The sequences were: K E K L E A P T L D Y D K Y F X E K A L G M R A X E I- for ArAT-I, and N L S D A L E M V N P S W I A K L F D L A Q G I E G I I S L- for ArAT-11. Taken together, these data indicate that

both enzymes are homodimers. They are therefore similar to ArAT enzymes previously reported from mesophilic species. For example, the TyrATs from E. coli and rat liver are both homodimers with subunit masses of 46 kDa [23] and 50 kDa [31], respectively. Although the N-terminal amino acid se- quences of these TyrATs have been reported 123, 311, there is no similarity between them and those of the two 7: litoralis enzymes. Initial characterization of the TyrATs from E. coli and rat liver were complicated by the occurrence of multiple active forms deriving from partial proteolysis of larger enzymes [23, 32, 331. These isoforms differed from their pre- cursors in the absence of N-terminal peptides. Therefore, in order to find stretches of amino acids similar to the amino- terminal sequences of ArAT-IT1 and ArAT-IIT1, the whole sequences of TyrAT from E. coli and rat liver were analyzed, but no similarities were found. Thus, notwithstanding the similarities at the level of the quaternary structure, aromatic aminotransferases from the archaeon 7: litoralis appear to differ significantly from their bacterial and eukaryotic coun- terparts at the level of the primary structure.

The pure preparations of ArAT-IT1 and ArAT-IIT1 were further analyzed by isoelectric focusing followed by silver staining. ArAT-ITl gave rise to three distinct bands (with PI values of 4.3, 4.5, 4.6), while a broad band, centered at pl 4.2, was observed for ArAT-IITI (data not shown). In both cases, the bands were positively stained by zymography using either phenylalanine, tryptophan or 3-iodo-tyrosine as the substrate, showing that each isoform was catalytically active. The structural differences between these isozymes are not known but the Edman degradation results suggest they do not arise from differences in N-terminal sequences.

Catalytic properties The enzymic activities of ArAT-ITl and ArAT-117'1 were

measured at 78 "C at various substrate concentrations in order to calculate the K,,, and k,,, values for the amino acid sub-

547

Table 3. Kinetic parameters of the aromatic aminotransferases isolated from Z litoralis. K,,, and k,,, for ArAT-I and ArAT-I1 from 1: litoralis were determined at 78°C in 0.2 M potassium phosphate pH 7.6 containing 62.5 pM pyridoxamine-P. For 2-oxoglutarate, 49mM phenylalanine was used as the amino donor, and for the amino acids, 4.7 mM 2-oxoglutarate was used as the amino acceptor.

Substrate K,, for k,.,K,, for - - ~ ~~~ - -

ArAT-I ArAT-I1 ArAT-I ArAT-I1

mM s - ' mM-' -

- - 2-Oxoglutarate 0.44 0.49 Phenylalanine 0.55 1.55 362.5 408.8 Tyrosine 2.39 2.37 72.4 210.0 Tryptophan 1.47 4.62 40.7 157.0

0.25

0 .20

8 0.15 K m 0 v)

e 9 0.10

0.05

0 I I I

260 305 350 395 440 485 5

Wavelength (nm)

Fig.3. Absorption spectra of ArAT-I1 from 1: litoralis. Spectra were acquired at a protein concentration of 1 mglml i n 50 mM Tris/ HC1 pH8.5 at 25°C. Curve 1 (-) ArAT-11; curve 2 (----) ArAT-I1 after the addition of 10 mM cysteine sulphinate; curve 3 (. . . I . .) ArAT-I1 after the consecutive addition of 10 mM cysteine sulphinate and 0.22 mM 2-oxoglutarate.

strates, phenylalanine, tyrosine and tryptophan, and for 2-oxoglutarate. The data are summarized in Table 3. Both enzymes had comparable affinities for the 0x0 acid, and a higher affinity for phenylalanine than for the other two amino acids and with similar k,,,lK,,, ratios. From these data it would appear that the two enzymes are equally efficient at transami- nating phenylalanine, but that tyrosine and tryptophan are preferentially utilized by ArAT-I1 rather than ArAT-I.

Primary plots of the reciprocal of phenylalanine transam- inase activity versus the reciprocal of substrate concentration were parallel (data not shown), which is expected for en- zymes with a two-step transfer catalytic mechanism. This suggests that the ArATs from 7: litoralis shuttle between a pyridoxal and pyridoxamine form during catalysis, as do most pyridoxal-5'-phosphate dependent aminotransferases (see [24, 251). To support this hypothesis, ArAT-IT1 (1 mg/ ml in 50 mM Tris/HCl pH 8.5) was titrated by addition of 1 O m M cysteine sulphinic acid, an a-amino acid analogous to aspartic acid, which spontaneously eliminates sulfite after transamination. Although cysteine sulphinate was a poor substrate for ArAT-IIT1, it promoted the irreversible shift of

Table 4. Activity of ArAT-IT1 and ArAT-IIT1 towards different amino acids. Activities were determined by assaying the purified enzymes at 78°C in 200 mM potassium phosphate pH 7.6, contain- ing 4.7 mM 2-oxoglutarate, 62.5 pM pyridoxamine-P and either phenylalanine (49 mM), tyrosine (12 mM), tryptophan (66 mM), al- mine (120 mM), aspartate (120 mM), valine (120 mM) or isoleucine (120 mM).

Amino acid Relative activity

ArAT-I ArAT-I1

%

Phenylalanine Tyrosine Tryptophan Alanine Aspart ate Val i ne Isoleucine

100 82 44 56 29 100 0.8 0.2 e o . l 0.1 0.4 eo.1 'eo.1 e o . l

- 300

250 -

v)

-200 z + 5

15C 2 - -100

i s 35 45 55 65 75' 85 95 1c5 1 I t .

Temperature ('C)

Fig. 4. Effect of temperature on the activity of ArAT-I and ArAT- I1 from 1: litoralis. The activities of ArAT-I (X) and ArAT-I1 (W) from ?: Zitoralis were determined in the temperature range 30- 105 "C, in the presence of saturating concentrations of phenylalanine and 2-oxoglutarate. The insert shows the Arrhenius plots of the k,,, values in the range 30-95°C.

the enzyme from the pyridoxal form, characterized by a maximum at 378 nm in the absorption spectrum, to the pyri- doxamine form. This is characterized by an absorption maxi- mum at 320 nm (Fig. 3). Upon addition of 2-oxoglutarate (0.22mM) to ArAT-IITl in the pyridoxamine form, the se- cond catalysis step took place and the pyridoxal form of the enzyme was restored (Fig. 3), thus confirming the two-step mechanism.

The ability of the two ArATs from T litoralis to transami- nate some non-aromatic amino acids is illustrated in Table 4. These were poor substrates and therefore high concentrations (120 mM) were required to measure significant activity. Whereas both enzymes efficiently catalyze the transamina- tion of the three aromatic amino acids with minor differences between phenylalanine, tyrosine and tryptophan, they are vir- tually inactive (< 1 %) towards branched-chain amino acids, aspartic acid and alanine.

The dependence of k,,, on temperature for ArAT-ITl and ArAT-IITZ is shown in Fig. 4. Over the 5-min assay period, both enzymes had barely detectable activity at 30°C and

548

Table 5. Comparison of aromatic aminotransferases from the archaea E litoralis and M. aeolicus. Data for M. aeolicus is taken from [20]. When measuring the K,, for 2-oxoglutarate phenylalanine was the amino group donor.

Parameter Value for

7: IitoruliJ M. ueolicus

ArAT-I ArAT-I1 ArAT-I ArAT-I1 ~~~

Molecular mass (kDa) 110 92 1.50 90

K,, Phe (mM) 0.55 1.55 0.4 1.2 K, 5 r (mM) 2.39 2.37 1.0 2.1 Km Trp (mM) 1.47 4.62 1.0 1.9 K, 2-oxoglutarate (mM) 0.44 0.49 0.6 0.6

Isoelectic point 4.3, 4.5, 4.6 4.2 4.4 4.9

showed maximum activity in the range of 95-100°C, which corresponds to the upper growth temperature of the organ- ism. The corresponding Arrhenius plots are also shown in Fig. 4. Neither enzyme showed a transition point over the temperature range 30- 95 "C. This was somewhat surprising as the limited number of hyperthermophilic enzymes purified so far typically show break points in the range 60-80°C (see [4]). For example, glutamate dehydrogenase from Pyrococ- cus furiosus, an organism that grows optimally at 100°C, exhibits a temperature-induced conformational change near 60°C which is coincident with the onset of catalytic activity [34]. From the data shown in Fig. 4, an activation energy of 67 kJ/mol was calculated for phenylalanine transamination by ArAT-IT1 and 38 kJ/mol by ArAT-IITL. This compares with a value of 46 kJ/mol reported for the AspAT from Sulfo- lobus solfataricus [35]. The difference in the slopes of the Arrhenius plots for the two 7: litorulis enzymes (Fig. 4) is also surprising since they are catalyzing the same reaction and have very similar kinetic properties (Table 3).

Relationship and function of aromatic aminotransferases from Archaea

The results described above show that two similar yet distinct enzymes (ArAT-IT1 and ArAT-IITI) account for the majority and probably all of the aromatic amino acid amino- transferase activity detected in cell-free extracts of 7: titor- alis. Two ArATs have also been identified in the methano- genic, mesophilic archaeon, M . aeolicus by Xing and Whit- man [20]. Termed ArAT-IMu and ArAT-IIMa, these were par- tially purified and characterized [20]. Like the two ArATs from 7: litoralis, they were active on phenylalanine, tyrosine and tryptophan, and almost inactive on branched chain amino acids, alanine and aspartic acid. These archaeal enzymes therefore differ from TyrAT and AspAT purified from E. coli, as both of these utilize both aromatic amino acids and aspar- tic acid as substrates [17, 231.

In addition, each of the two types of ArATs found in 7: litoralis had very similar physical and kinetic properties to the analogous enzyme in M. aeolicus (see Table 5). That is, the same characteristics distinguish the larger isoenzyme from the smaller isoenzyme in each organism. Interestingly, the K,,, values of the ArAT-I-type enzyme for aromatic amino acids are lower than those of the ArAT-11-type, whereas the K,,, values for 2-oxoglutarate are similar. This suggests that the two ArATs present may play different physiological roles. However, these two archaea have very different growth char-

acteristics. M . aeolicus grows autotrophically whereas T. li- toralis is an obligate heterotroph which utilizes only peptides as a nitrogen source [16] although certain carbohydrates are used as additional carbon sources [lS, 361. Hence, the ArATs in M. aeolicus were proposed to function in amino acid bio- synthesis, whereas this would not appear to be an absolute requirement in 7: litoralis. It is therefore puzzling that these two very different organisms should have such similar en- zymes for the transamination of aromatic amino acids.

Finally, we turn to the role of ArATs in 7: litoralis in light of its proteolytic-type metabolism. In the catabolic mode, the products of the ArAT reaction are glutamate and the trans- aminated form of the aromatic amino acids, the correspond- ing aryl pyruvate. 7: litoralis contains high intracellular con- centrations of an NADP-dependent glutamate dehydrogenase 1371, which uses glutamate to generate NADPH and 2-0x0- glutarate for biosynthetic purposes. This organism also pos- sesses significant concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase. This catalyzes the CoASH-dependent oxidative decarboxylation of indolepyru- vate, phenylpyruvate and p-hydroxypyruvate to the corre- sponding aryl acyl-CoA using 7: litoralis ferredoxin [38] as the electron acceptor (Mai, X. and Adams, M. W. W., unpub- lished data: see 14, 81). Reduced ferredoxin is thought to be oxidized by 7: litoralis hydrogenase leading to H, production (see [39]) but the fate of the aryl acyl-CoA derivatives is not known. It should be noted that in contrast to hyperthermophi- lic archaea such as 7: litoralis, bacteria that use aromatic acids as a carbon source contain decarboxylase-type enzymes which convert the aromatic pyruvates to the corresponding aldehyde rather than to the aryl acyl-CoA (see [40]). In any event, it is apparent that indolepyruvate ferredoxin oxidore- ductase uses the products of the ArAT reactions. Of course, it remains to be established whether ArAT-IT1 or ArAT-IIT1 (or both) function in a catabolic mode, and investigations to address this question are underway.

This work was supported by the Minister0 dell'Universita e Ricercu Scientificu, the Consiglio Nuzionale delle Ricerche (Pro- getto Finalizzuto Biotecnologie e Biostrumentazione), the Commis- sion of the European Communities, Human Capital and Mob Programme (contract ERB4050PL922141 to GS), the US Office of Naval Research (N00014-90-J-1894 to MWWA) and by the National Science Foundation (BCS-9320069 to MWWA). Ms M. E. Lisboa's skilful assistance in preparing the manuscript is gratefully acknowl- edged.

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