The Molecular Structure of Hyperthermostable AromaticAminotransferase with Novel Substrate Specificity fromPyrococcus horikoshii*

(Received for publication, August 10, 1999, and in revised form, October 14, 1999)

Ikuo Matsui‡§, Eriko Matsui‡, Yukihiro Sakai‡, Hisasi Kikuchi¶, Yutaka Kawarabayasi‡¶,Hideaki Urai, Shin-ichi Kawaguchii, Seiki Kuramitsui, and Kazuaki Harata‡§

From the ‡National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305,the ¶National Institute of Technology and Evaluation, Ministry of International Trade and Industry, Nishihara,Shibuyaku, Tokyo, and the iDepartment of Biology, Graduate School of Science, Osaka University, Toyonaka,Osaka 560-0043, Japan

Aromatic amino acid aminotransferase (ArATPh),which has a melting temperature of 120 °C, is one of themost thermostable aminotransferases yet to be discov-ered. The crystal structure of this aminotransferasefrom the hyperthermophilic archaeon Pyrococcus hori-koshii was determined to a resolution of 2.1 Å. ArATPhhas a homodimer structure in which each subunit iscomposed of two domains, in a manner similar to otherwell characterized aminotransferases. By the leastsquare fit after superposing on a mesophilic ArAT, theArATPh molecule exhibits a large deviation of the mainchain coordinates, three shortened a-helices, an elon-gated loop connecting two domains, and a long looptransformed from an a-helix, which are all factors thatare likely to contribute to its hyperthermostability. Thepyridine ring of the cofactor pyridoxal 5*-phosphate co-valently binding to Lys233 is stacked parallel to F121 onone side and interacts with the geminal dimethyl-CH/pgroups of Val201 on the other side. This tight stackingagainst the pyridine ring probably contributes to thehyperthermostability of ArATPh. Compared with otherArATs, ArATPh has a novel substrate specificity, theorder of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activityagainst Asp is due to lack of an arginine residue corre-sponding to Arg292* (where the asterisk indicates thatthis is a residues supplied by the other subunit of thedimer) in pig cytosolic aspartate aminotransferase. Theenzyme recognizes the aromatic substrate by hydropho-bic interaction with aromatic rings (Phe121 and Tyr59*)and probably recognizes acidic substrates by a hydro-philic interaction involving a hydrogen bond networkwith Thr264*.

Aminotransferases have been widely applied in the largescale biosynthesis of unnatural amino acids, which are in in-creasing demand by the pharmaceutical industry for peptido-mimic and other single-enantiomer drugs (1). These enzymeshave been classified into four families (I–IV) (2). Family I

includes the aspartate aminotransferases (AspATs),1 aromaticamino acid aminotransferases (ArATs), alanine ATs, and his-tidinol phosphate aminotransferase. All members of Family Iefficiently utilize a-ketoglutarate as an amino donor and glu-tamate as an amino acceptor. Eleven residues are invariantamong the enzymes belonging to Family I (2). The members ofFamily I are further subdivided into three subfamilies accord-ing to their amino acid sequence alignment (2, 3). Subfamily Iacomprises AspATs isolated from Escherichia coli, yeast,chicken, pig, and other organisms, and ArATs from prokaryotes(E. coli and Paracoccus denitrificans). In this subfamily, anarginine residue (Arg292*, according to the numbering for pigcytosolic AspAT (cAspATp) (4))2 is conserved. The Arg292* res-idue interacts with the v-carboxyl moiety of the dicarboxylicsubstrates (5–7). The arginine residue was not found in allmembers of subfamily Ig, despite the normally high degree ofconservation in active site residues (2, 8, 9). Subfamily Ib isspecialized for histidine biosynthesis (2, 4).

Recently, much research effort has been directed toward theisolation and characterization of enzymes from hyperthermo-philic archaea. Interest in these enzymes has increased, be-cause of their biotechnological potentials for novel application(10, 11) and because of the need for a better understanding oftheir intrinsic resistance to heat and denaturing processes. Themechanisms of their stability continue to be challenging andunresolved problems in biochemistry and biotechnology (10–13). An aspartate aminotransferase gene homolog (open read-ing frame identification number 1371) was identified viagenome sequencing in the hyperthermophilic archaeonPyrococcus horikoshii (14, 15). The gene (ArATPh) was ex-pressed in E. coli, the product was purified to homogeneity, andthe enzyme ArATPh was determined to be an aromatic amino-transferase belonging to subfamily Ig. We present the firstreport of the molecular structure of hyperthermophilic ArAT,which is an essential step in the effort to comprehend itsstabilizing mechanisms. We also discuss its novel substratespecificity and dual substrate binding mechanism for bothacidic and aromatic amino acids on the basis of its active sitestructure.

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The atomic coordinates and structure factors (code 1DJU) have beendeposited in the Protein Data Bank, Brookhaven National Laboratory,Upton, NY.

§ To whom correspondence should be addressed. For I. M., Tel.:81-298-546142; Fax: 81-298-546151 and for K. H., Tel.: 81-298-546194;Fax: 81-298-546194.

1 The abbreviations used are: AspAT, aspartate aminotransferase;AT, aminotransferase; ArATPh, aromatic amino acid AT from P. hori-koshii; cAspATp, pig cytosolic AspAT; AspATEc, E. coli AspAT;ArATEc, E. coli ArAT; ArATPd, P. denitrificans ArAT; PLP, pyridoxal59-phosphate; KetoPhe, phenylpyruvate; 2OG, 2-oxoglutaric acid;DTNB, 5,59-dithiobis (2-nitrobenzoic acid); MES, 2-(N-morpholino)eth-anesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.

2 Asterisks after the residue number indicate the residues suppliedby the other subunit of the dimer.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 7, Issue of February 18, pp. 4871–4879, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 4871

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MATERIALS AND METHODS

Chemicals—The pET-11a vector and ultracompetent E. coli XL2-Blue MRF9 cell were purchased from Stratagene (La Jolla, CA). ThepET-15b vector and E. coli strain BL21 (DE3) were obtained fromNovagen (Madison, WI). Vent DNA polymerase was purchased fromNew England Biolabs (Beverly, MA). Restriction enzymes were pur-chased from Promega and Toyobo (Osaka, Japan) and were usedaccording to the manufacturer’s recommendations. Ultrapure dNTPsolution was obtained from Amersham Pharmacia Biotech. L-Cyste-insulfinic acid, bovine DNase I, b-NADH, and malate dehydrogenasefrom porcine heart (mitochondrial) were purchased from Sigma.2-Oxoglutaric acid monosodium salt and DTNB were purchased fromNacalai Tesque (Kyoto, Japan). Isopropyl-b-D-thiogalactopyranosidewas purchased from Takara Shuzo (Otsu, Shiga, Japan).

Cloning of Genes and Construction of Expression Vector—The com-plete genome sequence of P. horikoshii has been reported by Kawaraba-yasi et al. (14, 15). Standard cloning techniques were used throughout.The aromatic aminotransferase (ArATPh) gene was amplified usingpolymerase chain reaction with primers having NdeI and BamHI re-striction sites according to a method reported previously (16). The se-quences of the primers were 59-TTTTGTCGACTTACATATGGCGCTA-AGTGACAGA-39 (underlining indicates the upper primer containingthe NdeI site) and 59-TTTTGGTACCTTTGGATCCTTAACCAAGGATT-TAAACTAG-39 (underlining indicates the lower primer containing theBamHI site). The amplified gene was digested by NdeI and BamHI, andthe digested fragment coding for ArATPh was inserted in an expressionvector pET-11a cut with the same restriction enzymes. The nucleotidesequence of the inserted gene was verified by sequencing on an AppliedBiosystems 373A DNA sequencer (Taq DyeDeoxy Terminator CycleSequencing Kit, Perkin-Elmer).

Overexpression and Purification of Recombinant Protein—The clonedgene was expressed using the pET-11a vector system in the host E. colistrain BL21 (DE3) according to the manufacturer’s instructions. Thehost cells were transformed with the constructed pET-11a/ArATPhplasmid, after which the production of the protein was performed ac-cording to the method described previously (16). The concentration ofthe expressed protein was determined using a Coomassie protein assayreagent (Pierce) and utilizing bovine serum albumin as the standardprotein. The crude enzyme solution was prepared from the transfor-mant E. coli, and the enzyme was purified using chromatography in aHiTrap Q column (Amersham Pharmacia Biotech) and a HiLoad Su-perdex 200 column (Amersham Pharmacia Biotech) (16). The purity ofthe enzyme samples was analyzed using SDS-polyacrylamide gel elec-trophoresis (17) and isoelectric focusing using a PhastSystem (Amer-sham Pharmacia Biotech). Protein sequencing of recombinant ArATPhwas performed by Takara Shuzo Co. Ltd. (Otsu, Shiga, Japan) using aprotein sequencer PSQ-1 (Shimazu, Japan).

Pre-steady-state Kinetic Studies of Half-transamination Reactions—Aliphatic amino acid substrates with unbranched side chains were usedto estimate the hydrophobic substrate specificities of the aminotrans-ferase. We used L-form amino acids for the sC3-sC6 substrates andDL-isoforms for the sC7-sC9 substrates (18); the aminotransferasetested here cannot use D-form amino acids as substrates. All measure-ments were carried out at pH 8.0 and 25 °C. The buffer solution con-tained 50 mM HEPES with 100 mM KCl and 10 mM EDTA.

The slow reaction was followed spectrophotometrically by monitoringthe change in absorption of the bound coenzyme at 380 nm. When thekapp value was directly proportional to the substrate concentration, thekmax/Kd value was calculated from the following equation (18, 19).

kapp 5 ~kmax/Kd!@S# (Eq. 1)

Thus, the catalytic efficiency, kmax/Kd, was given by kapp/[S] for thesesubstrates. The rapid reactions were followed using stopped flow spec-trophotometers from Union Giken (model RA-401) or Applied Photo-physics (model SX-17MW). The reaction curves conformed to a single-exponential process. The free energy differences (DGT

Þ) between thetransition state and unbound enzyme plus substrate for various sub-strates were calculated using the following equation (19, 20).

DGTÞ 5 RT~ln~kBT/h! 2 ln~kmax/Kd!! (Eq. 2)

where R is the gas constant, T is the absolute temperature, kB is theBoltzmann constant, and h is the Planck constant.

Temperature Dependence of Activity for Overall Transamination Re-action—The overall transamination reaction for the acidic substrateaspartate was measured spectrophotometrically at 340 nm using acoupled assay with malate dehydrogenase and NADH at pH 8.0 and

25 °C (20, 21), and the steady-state kinetics parameters, Km and kcat,were determined. The reaction mixtures contained 50 mM HEPES, 100mM KCl, 0.01 mM EDTA, 0.1 mM NADH, 2.5 units/ml malate dehydro-genase, 1 mM ArATPh, and various concentrations of L-aspartate or2-oxoglutarate. The activity of the hydrophobic substrate phenylalaninewas measured at pH 8.0 and 25 °C. The product formation of thephenylpyruvate was monitored at 280 nm using the molar extinctioncoefficient difference of 450 cm21 M21 between phenylpyruvate andphenylalanine (22). The reaction mixture contained 50 mM HEPES, 100mM KCl, 20 nM ArATPh, and various concentrations of L-phenylalanineor 2-oxoglutarate.

To determine temperature dependence, the activities for five sets ofsubstrates, tryptophan-2-ketoglutarate (Trp-2OG), tryptophan-phe-nylpyruvate (Trp-KetoPhe), histidine-2-ketoglutarate (His-2OG), histi-dine-phenylpyruvate (His-KetoPhe), and glutamate-phenylpyruvate(Glu-KetoPhe) were measured at different temperatures (25–90 °C) atpH 8.0. Based on the following molar extinction coefficients of 2-oxo acidderivatives, the concentration of the enzymatic products were calcu-lated: at 310 nm, 24.5 and 3200 cm21 M21 for 2OG and 3-indolepyru-vate, respectively, and at 280 nm, 21 and 450 cm21 M21 for 2OG andKetoPhe (22), respectively. The product from His was monitored at 293nm using a coefficient difference of 3050 cm21 M21 obtained to subtractthe spectrum for the reaction of histidine with the PLP enzyme fromthat of the pyridoxamine 59-phosphate enzyme (23). The reaction mix-ture contained 50 mM HEPES, 100 mM KCl, 20 nM ArATPh, and variousconcentrations of amino acids or keto-acids.

Optimum Temperature and Thermostability for ArATPh Reaction—The optimum temperature for ArATPh activity was measured as de-scribed previously (24). The enzyme reaction was carried out in asolution (3.05 ml) containing ArATPh (3.8 nM), L-cysteinsulfinic acid(12.8 mM), 2-oxoglutaric acid (2.0 mM), EDTA (98 mM), and DTNB (1.5mM) in 50 mM phosphate buffer (pH 6.5) at 30–98 °C, and the rate ofincrease in absorbance at 412 nm because of the reduction of DTNB wasmonitored for 5 min. For controls, the reactions were performed underthe same conditions but without the enzyme.

To determine thermostability, the enzyme solutions (0.1 mg/ml) in 20mM phosphate buffer (pH 6.5) were incubated at 95 °C for 90 and 120min and then autoclaved in sealed Eppendorf tubes at 110 °C for 5, 15,30, and 90 min. The heated enzymes were assayed in duplicate at 90 °C,as described elsewhere (24).

Spectroscopy of Coenzyme—To investigate the ionization of the inter-nal Schiff base, the absorption spectra of the enzyme at a proteinconcentration of approximately 20 mM in a 1-cm cell were measured at25 °C using a Hitachi spectrophotometer (model U-3000). The buffersolution was comprised of 100 mM KCl, 0.01 mM EDTA, and a buffercomponent of 50 mM MES, 50 mM PIPES, or 50 mM HEPES.

pH Stability—The gross conformation and pH stability of ArATPhwere studied using CD spectroscopy. The CD spectra of ArATPh, at aprotein concentration of approximately 0.1 mg/ml in a 1-mm cell, weremeasured at 25 °C using a spectropolarimeter (J-720W, Jasco, Japan).The solution was comprised of 100 mM KCl and a buffer component of 50mM acetate, 20 mM phosphate, 50 mM borate, or 20 mM carbonate.

Scanning Calorimetry—The thermal denaturation curve of ArATPhwas measured using a Nano Differential Scanning Calorimeter(CSC5100, Calorimetry Science Co.). Before measurement, the enzymesolution (1 mg/ml) was dialyzed against 20 mM phosphate buffer, pH6.5, and degassed for 15 min using an aspirator. The sample cell wasfilled with the degassed enzyme solution, and the reference cell wasfilled with the outer solution from the dialysis. The measurement wasperformed at a temperature range of 0–125 °C. A scan rate of 1 K/minwas used throughout. The denaturation profile was analyzed usingNano differential scanning calorimetry CpCalc data analysis software(Calorimetry Science Co.).

Structure Determination, Refinement, and Model Building—Crystalswere obtained using the hanging drop vapor diffusion technique. Anequi-volume of 3 M 1,6-hexane-di-ol solution at pH 7.5 (100 mM HEPESbuffer) containing 10 mM MgCl2 was added to a protein solution con-taining 1.6% ArATPh and 20 mM pyridoxal-59-phosphate and a 10-mldroplet of the solution was equilibrated with 1 ml of a 3 M 1,6-hexane-di-ol solution. Crystals were grown at room temperature for 1 week.X-ray diffraction experiments were carried out on an Enraf FASTdifferactometer equipped with a FR571 generator (40 kV, 50 mA; focalspot size, 0.2 mm), and intensity data were collected at a resolution of2.1 Å for the native crystal and at 3.0 Å for the heavy atom derivatives.

The structure was determined using the multiple isomorphous re-placement method. A structure model was built on an electron densitymap calculated with multiple isomorphous replacement phases with afigure of merit of 0.93. The amino acid sequence was unambiguously

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traced on the map and most of the side chains were identified. Thestructure was refined to a resolution of 2.1 Å using X-PLOR (25). Allcoordinates have been deposited with the RCSB Protein Data Bank asentry 1DJU. In the substrate-binding model, the coordinates of ArATPhwith water molecules were fixed, but the torsion angles of the sub-strates were changed to find the best fitting configuration to theenzyme.

Sequence Alignment and Phylogenetic Tree—We performed a se-quence alignment of 11 aminotransferases within subfamily 1g usingthe GeneWorks program (IntelliGenetics, Inc., Mountain View, CA)based on a PAM-250 scoring matrix. The compared enzymes were asfollows: ArATPh, AspATs from thermophilic Bacillus sp. (26), Thermusthermophilus HB8 (8), Rhizobium meliloti (27), Bacillus subtilis (28),Methanococcus jannaschii (29), and Sulfolobus solfataricus (30); tyro-sine ATs from human (31), rat (32), and Trypanosoma cruzi (33); andalanine ATs from human (34), rat (35), and Panicum miliaceum (36).Phylogenetic trees for the same sequences were constructed using theGeneWorks program based on the unweighted pair group method withan arithmetic mean (37).

RESULTS

Sequence Alignment and Phylogenetic Tree of ArATPh—Be-cause we were unable to construct a united alignment amongaminotransferases belonging to the subfamilies 1a, 1b, and 1gfrom P. horikoshii, other archaea, bacteria, and eukaryotesbecause of a lack of similarity between each subfamily, wemade the alignment using 11 candidates belonging to subfam-ily 1g to identify its conserved residues. The best alignmentwas obtained with the five thermophilic aminotransferasesshown in Fig. 1. ArATPh showed poor identity to E. coli AspAT(AspATEc) (38), E. coli ArAT (ArATEc) (39, 40), and P. deni-trificans ArAT (ArATPd) (41), which are well known membersof subfamily Ia. According to these results, ArATPh was nom-inated to the aminotransferase subfamily Ig (2, 3). Further-more, ArATPh was closer to the thermophilic AspATs than tothe tyrosine ATs from eukaryotes in subfamily Ig.

Overexpression, Purification, and Oligomeric Structure of Re-combinant ArATPh—The ArATPh gene was abundantly ex-pressed in E. coli BL21 (DE3), and the recombinant ArATPhcomprised 30% of the total protein. After heat treatment at80 °C for 15 min, which removed most of the endogenous E. coliproteins, the protein was purified to homogeneity by sequentialchromatography on HiTrap Q and HiLoad Superdex 200 col-umns. The final preparation of the ArATPh displayed a singleband (42 kDa) on SDS-polyacrylamide gel electrophoresis. Iso-electric focusing indicated a pI value of 5.2 for ArATPh. TheN-terminal sequence of the recombinant ArATPh was ALS-DRLELVSASEIRKL, which was identical to that deduced fromthe DNA sequence without the initial methionine residue. Theenzyme had an apparent molecular mass of 56 kDa as esti-mated by gel filtration on a calibrated TSK gel G2000SWXLcolumn, and a subunit molecular mass of 42 kDa as estimatedby SDS-polyacrylamide gel electrophoresis. This suggests thatit has a dimeric structure similar to other aminotransferases(24, 34, 42–45).

Optimum Temperature of the Recombinant ArATPh—Theoptimum temperature of this enzyme was 90 °C, which repre-sents an extreme thermophilic characteristic. The kapp ofArATPh increased steadily in the range of the temperaturestudied here. The kapp for L-cysteinsulfinic acid and 2-ketoglu-taric acid as substrates was 1.39 3 102 s21 at 90 °C and pH 6.5.Like several other thermophilic enzymes (46–48), the recom-binant ArATPh shows a thermal transition in conformation asindicated in Arrhenius plots near 70 °C (data not shown).

Substrate Specificity—DGTÞ, the free energy difference be-

tween the unbound enzyme plus substrate (E 1 S) and thetransition state (ESÞ), was calculated for various substratesusing Equation 1 or 2. A smaller DGT

Þ value indicates higherenzyme activity. As shown in Table I, Tyr is the best substratehaving a kmax/Kd (M21 s21) value of 1.2 3 105. Three aromatic

amino acids (Tyr, Phe, and Trp) and Glu are good substrates forArATPh, whereas Asp is a poor substrate having a kmax/Kd

(M21 s21) value of 9.1. ArATPh showed moderate activity onHis. The activity of ArATPh toward a series of aliphatic sub-strates with straight side chains was enhanced as the sidechain length increased. The activity of ArATPh was maximalfor an 8-carbon substrate (2-amino octanoic acid).

Comparison of the Substrate Specificity for ArATs from Dif-ferent Origins—The steady-state kinetic parameters ofArATPh using an overall transamination reaction between2OG and Asp or between 2OG and Phe were measured at 25 °C(the upper section of Table II). The enzyme activity against Phewas high with a kcat/Km value of 5.2 3 103 M21 s21, but theactivity against Asp was very low (2.2 M21 s21). The differencebetween these kcat/Km values was on the order of 103, whereasthe difference for ArATEc (from E. coli) and ArATPd (from P.denitrificans) was approximately 10-fold. At the optimum re-action temperature of 90 °C, ArATPh has approximately a 102-

FIG. 1. Aligned amino acid sequences of five thermophilic ami-notransferases belonging to subfamily Ig. Thermus, T. thermophi-lus HB8 (8); Bac.sp, thermophilic Bacillus sp. YM-2 (26); Metha2, M.jannaschii isozyme 2 (29); Ph, P. horikoshii (present paper); Sulfo, S.solfataricus (30). The conserved residues, identified automatically bythe GeneWorks program, are shown in open boxes. Large capital lettersindicate the completely conserved residues among the 11 candidateslisted under “Materials and Methods.” The numbering is according tocAspATp (4). R292 indicates the position corresponding to Arg292* incAspATp.

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and 10-fold higher activity for His and Glu, respectively, thandoes ArATEc or ArATPd at 25 °C (Table II). This indicates thatGlu is the best acidic substrate for ArATPh.

Spectroscopic Properties of the Enzyme-bound CoenzymesPLP and Pyridoxamine 59-Phosphate—An internal Schiff baseis formed between Lys233 (corresponding to Lys258 in cAspATp)and the aldehyde group of the coenzyme PLP. The reactionproduces a spectral change in the visible absorption region.Changes in the apparent molar absorption coefficients for thePLP form enzyme at 420 and 370 nm were plotted against pH.The Schiff base pKa value was determined to be 5.1.

Three-dimensional Structure of ArATPh—The space group ofthe protein crystal is P212121, and the cell dimensions are a 564.01, b 5 124.87, and c 5 128.78 Å. The structure was solvedusing the multiple isomorphous replacement method at a res-olution of 3.0 Å using four heavy atom derivatives: K2PtCl4,methyl mercury chloride, p-chloromercuribenzenesulfonic acid,and mersalyl acid. These data are presented in Table III. Thestructure was refined at 2.1 Å resolution to the R value of 0.185and Rfree of 0.254, respectively. The root mean square devia-

tions of bond distances and angles from their ideal values were0.017 Å and 3.24°, respectively. The (f, c) values for all theamino acid residues except Thr264 fell in a normal region in theRamachandran plot (data not shown). The crystal having a Vm

5 2.9 Å3/Da contains two molecules related with local 2-foldsymmetry in an asymmetric unit. ArATPh has a dimer struc-ture (Fig. 2). One dimer molecule has two active sites, and eachactive site binds one PLP. In both subunits, the N-terminalregion of residues 2–11 form a short a-helix, but region 12–26is missing in the final structure model because no significantelectron density was observed in the 2Fo 2 Fc and Fo 2 Fc mapsfor the region (Fig. 3). The molecule consists of two domains.The large domain has a a/b structure comprised of six a-helices(H3–H8) and seven b-strands (S1–S7), as assigned by the pro-gram DSSP (49). The strands form a twisted sheet structure, onboth sides of which helices are arranged. The small domainconsists of three a-helices (H10–H12) and a b-strand (S8). Along a-helix (H10) links to the large domain via an a-helix (H9).

The molecular replacement method using the structuresAspATEc, ArATPd, and cAspATp as templates (6, 7, 50) wasnot successful for solving the ArATPh structure because of pooridentity of the primary sequences and a large deviation in themain chain coordinates. By the least square fit after superpos-ing ArATPh on ArATPd, only 295 pairs of corresponding aminoacid residues were present in the Ca-Ca distance less than 3 Å,and their root mean square deviation was 2.0 Å. Several struc-tural differences were observed between ArATPh and ArATPdas shown in Fig. 4. The 5th, 11th, and 13th a-helices of ArATPdare shorten by several amino acid residues in ArATPh, corre-sponding to H4, H8, and H10, respectively. Interestingly, the9th a-helix (from Tyr225 to Val250) in the ArATPd molecule istransformed into a long loop (from Tyr202 to Phe222) between S5and S6 of ArATPh. A loop (Val345–Ser364) between H11 and S8of the small domain of ArATPh covers one end of the cleftformed at the interface between the large and small domains,although the corresponding loop is shorter in the ArATPdmolecule and is unable to form a lid on the cleft.

As shown in Figs. 5 and 6, PLP is positioned at the bottom ofthe active site and forms an internal aldimine bond (Schiff baselinkage) with the catalytic residue Lys233 (corresponding toK258 in cAspATp). The phosphate moiety of PLP forms hydro-gen bonds with Oh of Tyr59* (Tyr70* in cAspATp), N of Ala96

(Thr109 in cAspATp), Og of Ser232 (Ala257 in cAspATp), and Nh1and Nh2 of Arg241 (Arg266 in cAspATp; Fig. 5A). The pyridinering of PLP interacts with both methyl groups of Val201 (Ala224

in cAspATp) on one side, and on the other side with the phenylring of Phe121 (Trp140 in cAspATp), by stacking interaction. The0–3 atom on the pyridine ring of PLP forms direct hydrogenbonds with the side chains of Asn171 (Asn194 in cAspATp) andTyr202 (Tyr225 in cAspATp). The N1 atom on the pyridine ringforms a hydrogen bond with the side chain of Asp199 (Asp222 incAspATp). Thus, PLP is fixed tightly at the bottom of the activecenter.

Gross Conformation and pH Stability—The gross conforma-tion and pH stability of ArATPh were studied using CD spec-troscopy at 25 °C. The CD spectrum in the region between 200and 250 nm exhibited double negative minima at 209 and 223,which are characteristic of an a-helical structure (data notshown). The a-helical content is estimated to be approximately40%, according to the method of Chen et al. (51). The enzyme isstable between pH 4 and 11 for 24 h at 25 °C.

Heat Stability—The residual ArATPh activity remaining af-ter heating was measured to determine the half-life of theenzyme at 95 and 110 °C. The half-life of ArATPh is 30 min at110 °C, and the enzyme is stable at 95 °C in 20 mM phosphatebuffer (pH 6.5).

TABLE IIComparison of the kinetic parameters (kcat/Km s21 M21) of the overall

transamination reactions at pH 8.0 among ArATs fromhyperthermophilic and mesophilic organisms

The reaction temperatures are shown in parentheses.

Substrate ArATPh (25 °C) ArATEc(25 °C)a

ArATPd(25 °C)b

Asp 2.2 6 0.1 3.7 3 104 9.4 3 104

Phe 5.2 6 0.3 3 103 9.6 3 105 2.2 3 105c

ArATPh (90 °C)

Trp 2.6 6 0.4 3 105 3.2 3 105 7.0 3 104

His 3.5 6 0.3 3 104 6.7 3 102c 3.4 3 102c

Glu 2.0 6 0.2 3 105 1.0 3 104c 2.1 3 104c

2-Ketoglutarate 2.6 6 0.3 3 105 2.7 3 105 7.1 3 105

Phenylpyruvate 1.1 6 0.2 3 107 2.0 3 107c 1.6 3 106

a Cited from Ref. 39.b Cited from Ref. 41.c The values were obtained by using the kinetic parameters for the

half-reactions.

TABLE IThe kmax/Kd values of ArATPh for the half-transamination reactions

with a series of substrates at pH 8.0 and 25 °CThe chain lengths of the straight substrate are shown in parentheses.

Substrate kmax/Kd DGTÞ

s21M

21 kcal/mol

Tyr 1.2 6 0.2 3 105 10.5 6 0.1Phe 3.8 6 0.4 3 104 11.2 6 0.1Trp 1.3 6 0.3 3 104 11.8 6 0.1His 2.2 6 0.3 3 103 12.9 6 0.1Met 4.9 6 0.9 3 10 15.1 6 0.1Leu 1.2 6 0.2 3 10 15.9 6 0.1Val 3.3 6 0.6 3 1022 19.4 6 0.1Ile 5.4 6 1.9 3 1023 20.5 6 0.2Glu 3.1 6 0.4 3 104 11.3 6 0.1Asp 9.1 6 1.9 16.1 6 0.1Asn 6.8 6 0.6 16.30 6 0.04Gln 1.1 6 0.3 17.4 6 0.1Ser 5.6 6 0.6 3 1021 19.1 6 0.1Thr 3.7 6 0.7 3 1023 20.7 6 0.1Arg 1.6 6 0.5 3 1022 19.9 6 0.2Lys 9.9 6 1.6 3 1023 20.1 6 0.1Ala (sC3) 1.9 6 0.3 17.0 6 0.12-amino butylic acid (sC4) 2.1 6 0.4 17.0 6 0.1n-Val (sC5) 5.8 6 0.7 16.4 6 0.1n-Leu (sC6) 1.9 6 0.3 3 10 15.7 6 0.12-amino heptanoic acid (sC7) 1.3 6 0.2 3 102 14.5 6 0.12-amino octanoic acid (sC8) 1.3 6 0.1 3 103 13.20 6 0.032-amino nonanoic acid (sC9) 8.6 6 1.8 3 102 13.4 6 0.1

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The heat capacity change of ArATPh was measured usingdifferential scanning calorimetry from 0 to 125 °C at pH 6.5.The heat capacity change was only observed during the firstscan in the differential scanning calorimetry measurement,indicating that the heat denaturation profile of ArATPh is dueto an irreversible denaturation process. The profile showed onemajor peak at 120.1 °C. The molar enthalpy change, DH, wascalculated to be 2.4 3 103 kJ/mol for the homodimer.

DISCUSSION

Structural Elements Providing Hyperthermostability onArATPh—ArATPh is one of the most thermostable aminotrans-ferases ever to be purified (52), having a melting temperatureof 120 °C. ArATPh is a homodimer in which each subunit isconstituted of two domains, similar to other well characterizedaminotransferases, such as AspATEc, ArATPd, and cAspATp(6, 7, 50). However, its structure could not be solved by themolecular replacement method because of a poor sequence sim-ilarity, large deviation in the main chain coordinates, and localchanges in the secondary structure, including three shorteneda-helices and a long loop transformed from an a-helix (Fig. 4).Another unique characteristic is its elongated loop betweenH11 and S8 (Figs. 3 and 4). The loop intimately connects twodomains and covers one end of the cleft formed at the interfaceof the two domains. Because numerous hydrophobic residueswere observed inside the cleft, the elongated loop, which closelybinds the two domains and acts like a lid to shield the cleft fromsolvents, might be one of the factors that account for the hy-perthermostability of ArATPh.

On the basis of the ArATPh structures, the surface area forone amino acid residue was calculated by dividing the accessi-ble surface area of the dimer by total residue numbers toevaluate molecular compactness. The values of ArATPh andArATPd are 28.0 and 33.6 Å3, respectively. The lower surfacearea for one residue of ArATPh molecule might be due to tightpacking of the polypeptide chain into the homodimer structure.Another prominent difference in ArATPh is the large numberof charged residues (Asp, Glu, Lys, and Arg) on its molecularsurface compared with the ArATPd. The occupancy of thecharged residues in the accessible surface area of ArATPh andArATPd molecules are 73.3 and 48.1%, respectively. On thecontrary, the frequency of polar contacts less than 3.3 Å, in-cluding hydrogen bond and ion pair among these charged res-idues on the surface is decreased to 36.0% for ArATPh incomparison with the value, 51.3%, of ArATPd. The accessiblesurface of ArATPh has higher hydrophilicity with a lower num-ber of ion pairs than that of ArATPd. The compact packing andthe remarkably water-attractive surface of ArATPh are prob-ably major factors contributing to its hyperthermostability.

The PLP molecule of ArATPh is fixed tightly with nine hy-drogen bonds at the bottom of the active site cleft (Fig. 5A). Oneside of the pyridine ring of PLP interacts with the geminaldimethyl groups of Val201 (Ala224 in cAspATp), whereas theother side is stacked parallel with the phenyl ring of Phe121

(Trp140 in cAspATp). In AspATEc, the methyl group of Ala224

interacts with the pyridine ring of PLP on one side, and on theother side, the pyridine ring stacks to Trp140 with a 20° incli-nation angle. In the thermophilic enzymes of subfamily Ig,valine or isoleucine is found at the position corresponding toVal201 of ArATPh, whereas the residue is replaced with Ala inthe mesophilic enzymes of subfamily Ia (2, 3). The interactionof Ala with PLP should be weaker than those of V/I in thermo-philic aminotransferases because of the lack of a geminal di-methyl-CH/p interaction (53). In subfamily Ig of the thermo-philic archaea (Fig. 1), the phenyl ring of Phe or Tyr,corresponding to Phe121 in ArATPh, always stacks to the pyr-idine ring of PLP. In subfamily Ia from the mesophilic organ-isms and subfamily Ig from the thermophilic prokaryotes,these residues are replaced by tryptophan, which has a bulkierside chain with a wider surface area than does the phenyl ring(2). Consequently, a combination of the Phe121 and Val201 res-idues stacking tightly to the pyridine ring of PLP may contrib-ute to the hyperthermophilic properties of ArATPh. Furthercrystallographic studies are in progress to better understandthe mechanisms underlying the hyperthermostability of thisenzyme.

PLP-binding Structure of ArATPh—In the PLP molecule ofArATPh, the number of hydrogen bonds fixing the phosphate

TABLE IIISummary of data collection and phasing

Data collection NativeHeavy atom derivatives

K2PtCl4 Methyl HgCl PCMBSa Mersalylacid

Maximum resolution 2.1 3.0 3.0 3.0 3.0Measured data 359213 96566 103007 99825 93801Unique data 57422 20183 20871 20454 21244Completeness 0.905 0.945 0.978 0.958 0.995Rmerge 0.046 0.039 0.038 0.037 0.035

PhasingHeavy atom derivatives

K2PtCl4 Methyl HgCl PCMBS Mersalyl acid

Heavy atom sites 2 6 6 5RK 0.121 0.048 0.056 0.066RC 0.654 0.399 0.429 0.446Phasing power 0.63 5.15 4.17 3.34

a PCMBS, p-chloromercuribenzenesulfonic acid.

FIG. 2. The crystal structure of ArATPh. Ca tracing of ArATPhdimer. Subunits A and B are colored red and blue, respectively. ThePLP molecules are represented by a ball-and-stick model. The figurewas produced using the program Turbo-Frodo.

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moiety is reduced from six to five (Fig. 5A), because of replace-ment of the Ser255 residue, which is conserved in both AspATEcand ArATPd (6, 7). The large conformational change of thephosphate moiety of PLP is induced by a shift in the side chainsof Ala96, Ser232, and Arg241 from the corresponding residues inAspATEc. The phosphate moiety moves parallel to the plane ofthe pyridine ring of PLP, whereas the pyridine ring is con-served at the same position as in AspATEc. The movement ofthe phosphate moiety in the opposite direction might be apositive adjustment of the cofactor to compensate for changesin the secondary structure that account for its hyperthermo-stability. Interestingly, the Oh position of Tyr202 in ArATPh isalmost identical to that of the corresponding Tyr225 residue ofAspATEc (Fig. 5A), whereas the coordinates of the main chainparts in both Tyr residues are shifted by more than 2 Å. The

Tyr residues of both ArATPh and AspATEc are close enough toform a hydrogen bond with O3H of PLP. The position of thev-carboxyl of Asp199 forming a hydrogen bond with N1H of PLPis also identical to that of Asp222 of AspATEc. These resultsstrongly indicate that the pyridine ring must be fixed preciselyat the conserved position in the active center of ArATs to makethe cofactor fully active, although the phosphate moiety can bepositioned according to the steric requirements. The Oh of theTyr202 residue of ArATPh can also form a hydrogen bond withthe imino group of the Schiff base. The angle of the iminoproton on the C5N plane of ArATPh is sufficient to form ahydrogen bond with Tyr202; however, the corresponding anglein AspATEc seems less suitable to form a hydrogen bond withTyr225 (Fig. 5A). The pKa of the Schiff base of ArATPh wasdetermined to be 5.1, which is the lowest value ever reported;

FIG. 3. The folding topology of the monomer of ArATPh. A, a-helices, b-strands, and loops are colored red, blue, and yellow, respectively.The green a-helix corresponds to the N-terminal a-helix followed by the disordered region. The a-helices and b-strands of the a/b structure arenumbered from the N terminus. The PLP molecules are represented by a space-filling model. The figure produced using the program Turbo-Frodo.B, topology diagram of ArATPh. a-Helices are shown as cylinders (red), b-strands are arrows (green), and the numbering is the same as that in A.The sequential numbers of the first and last residues in each secondary structure element are indicated.

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FIG. 5. The PLP binding structure of ArATPh. A, the stereoview for the superposition of ArATPh (red) and AspATEc (blue) by the PLPfitting. Although the overall structure of AspATEc including the PLP binding profile (6) is quite similar to that of ArATPd (7), the AspATEc wasselected as a reference structure for the superposition because of its general popularity historically. The residue numbers indicate the positions inthe ArATPh molecule (red). Dotted lines indicate the hydrogen bonds in ArATPh (red). B, nomenclature of atoms for PLP.

FIG. 4. Superimposition of ArATPh (green) on ArATPd (red) (7). The figure was produced using the program Turbo-Frodo. The PLPmolecule of ArATPh is represented by a space-filling model.

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the pKa values of AspATEc, T. thermophilus AspAT, andArATEc were reported to be 6.8 (54), 6.1 (8), and 6.65 (55),respectively. This low pKa value is probably due to rotation ofthe C5N plane of the Schiff base against the pyridine ring ofPLP to control hydrogen bonding between the imino group andthe Tyr202 residue and may also be due to the unique environ-ment around the PLP molecule caused by changes in the resi-dues stacked to PLP (Fig. 5A).

Active Site Structure and Substrate Binding Models—Theactive site structure with the best substrate, Tyr, is shown inFig. 6. The a-carboxylate of Tyr was fixed at the active site bytwo salt bridges with Arg362 (corresponding to R386 incAspATp) and three hydrogen bonds with Gly34, Asn171, andTyr320. The phenyl ring of Tyr and the aromatic group of Phe121

undergo an energetically favorable “edge-to-face” interaction(56), and the aromatic ring of Tyr59* is located very closely, butnot in parallel, to the phenyl ring of Tyr. Thus, the best sub-strate can be trapped in the hydrophobic pocket formed byPhe121, Tyr59*, the pyridine ring of PLP, Met260* (correspond-ing to Arg292* in cAspATp), and Val122 (Glu141 in cAspATp). Inthis binding model, the OH group of Tyr is located at a distancesufficient to form hydrogen bonds with Og1 of Thr264* (Ser296*in cAspATp) and with the phosphate moiety of PLP. The inter-nal aldimine bond between PLP and Lys233 (corresponding toLys258 in cAspATp) is located so close that a new externalaldimine bond can be formed between PLP and the a-aminogroup of Tyr.

Another binding model was formed with Glu, one of the bestacidic substrates. A water molecule (Xaa126*) is present at thecenter of three adjacent groups: the g-carboxyl of Glu, theOg1H of Thr264*, and the phosphate residue of PLP. The prox-imity (within 3 Å) of the water molecule and the adjacentresidues allows formation of a hydrogen-bond network amongthem. The water molecule (Xaa126*) may be important in bind-ing Glu to the active center, as indicated by the reportedlycomplex structure of ArATPd with maleate (7). Furthermore,the g-carboxyl group of Glu is parallel to the phenyl ring ofTyr59*, suggesting a van der Waals’ interaction between thetwo groups. This sort of weak interaction may be important forthe recognition of C5 substrates (Glu and 2OG) in amino acidaminotransferases, because the Y70*S mutant of AspATEc is

reportedly less active against these substrates (57). The phenylring at position 70 is essential for the recognition of the Glu-2OG pair as substrates. Hence, in the binding model ofArATPh, both ends of the acidic substrate, Glu, are fixed at theactive center of the enzyme by three major interactions: 1) saltbridges between Arg362 and the a-carboxylate of Glu, 2) twohydrogen bonds located between Gly34 and the a-carboxylate,and between Gly34 and a-amino groups of the substrate, and 3)the capturing of the g-carboxylate by the hydrogen bond net-work through the water molecule Xaa126* and by a weak inter-action with Tyr59*. The low activity against Asp is explained intwo ways: 1) the lack of an arginine residue corresponding toArg292* of cAspATp, which interacts with the distal carboxylateof the acidic substrate and 2) the lack of a hydrogen bondnetwork through the water molecule Xaa126* and no interac-tion with Tyr59*, because of a lack of one methylene unit at theg position.

Substrate Specificity of ArATPh—As shown in Tables I andII, ArATPh prefers the substrates in the following order inkmax/Kd: Tyr . Phe . Glu . Trp . His .. Met . Leu . Asp .Asn. The substrate specificity differs from those of the meso-philic ArATs, including ArATPd, with the preference in kcat/Km

being Tyr . Phe 5 Asp . Trp . Glu (41). Thermostable ArATsfrom Pyrococcus furiosus and Methanococcus aeolicus were alsoreported to have distinct substrate specificity in kcat/Km: Phe .Trp . Tyr (52, 58). Consequently, ArATPh has a novel sub-strate specificity compared with other ArATs.

Aminotransferases are increasingly applied to the large scalesynthesis of unnatural and nonproteinogenic amino acids (1).Typically exhibiting relaxed substrate specificity, rapid reac-tion rates, and no need for cofactor regeneration, they possessmany characteristics that make them useful for biocatalysis.Because of its novel substrate specificity and high level ofresistance to organic solvents (data not shown), ArATPh willcontinue to be a useful biocatalysis for the synthesis of unnat-ural compounds.

Acknowledgments—We thank Miyuki Ishimura for assistance withthe differential scanning calorimetry analysis and valuable discussions.Koichi Honda is gratefully acknowledged for his practical advice andvaluable discussions.

FIG. 6. The substrate-binding model for ArATPh represented by stereoview. The front view of the active site with Tyr. The modelstructure is presented in a sphere with a 15-Å radius surrounding the substrate. The subunits A and B are colored green and pink, respectively.The substrates and PLP are colored blue and red, respectively. The possible hydrogen bonds are represented as dashed lines. The yellow figure,3.4, indicates the distance (Å) between the internal aldimine bond and the a-amino group of Tyr.

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Hideaki Ura, Shin-ichi Kawaguchi, Seiki Kuramitsu and Kazuaki HarataIkuo Matsui, Eriko Matsui, Yukihiro Sakai, Hisasi Kikuchi, Yutaka Kawarabayasi,

Pyrococcus horikoshiiNovel Substrate Specificity from The Molecular Structure of Hyperthermostable Aromatic Aminotransferase with

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