Characterization of a Thermostable L-Arabinose (D-Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilicbacterium Thermotoga maritima was cloned and overexpressed inEscherichia coli as a fusion protein containing a C-terminalhexahistidine sequence. This gene encodes a 497-amino-acid proteinwith a calculated molecular weight of 56,658. The recombinantenzyme was purified to homogeneity by heat precipitation followedby Ni²⁺ affinity chromatography. The native enzyme was estimatedby gel filtration chromatography to be a homotetramer with amolecular mass of 232 kDa. The purified recombinant enzyme hadan isoelectric point of 5.7 and exhibited maximal activity at90°C and pH 7.5 under the assay conditions used. Its apparentK_m values for L-arabinose and D-galactose were 31 and 60 mM,respectively; the apparent V_max values (at 90°C) were 41.3U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalyticefficiencies (k_cat/K_m) of the enzyme were 74.8 mM^-1 ·min^-1 (L-arabinose) and 8.5 mM^-1 · min^-1 (D-galactose).Although the T. maritima AI exhibited high levels of amino acidsequence similarity (>70%) to other heat-labile mesophilicAIs, it had greater thermostability and higher catalytic efficiencythan its mesophilic counterparts at elevated temperatures. Inaddition, it was more thermostable in the presence of Mn²⁺ and/orCo²⁺ than in the absence of these ions. The enzyme carried outthe isomerization of D-galactose to D-tagatose with a conversionyield of 56% for 6 h at 80°C.

INTRODUCTION

Top

Introduction

Of the eubacteria whose genomes have been sequenced to date,Thermotoga maritima has the highest percentage of genes thatare most similar to archaeal genes (20). This bacterium is alsoof evolutionary importance, because small-subunit rRNA phylogenyhas shown that it is one of the deepest and most slowly evolvinglineages of the eubacteria. Thus, it could be useful for elucidatingthe evolutionary relationship between thermophilic eubacteriaand archaea. In addition, it metabolizes many simple and complexcarbohydrates, including glucose, sucrose, starch, cellulose,and xylan (9). Thermostable enzymes from T. maritima involvedin carbohydrate metabolism are very attractive for industrialapplications (4, 15, 24).

Almost 7% of the predicted coding sequences in the T. maritimagenome are involved in metabolism of simple and complex sugars(20). Several genes encode proteins involved in arabinose metabolism.Among them, the araA gene encoding L-arabinose isomerase (AI)(EC 5.3.1.4), which catalyzes the conversion of L-arabinoseto L-ribulose, not only is important for pentose sugar isomerizationin vivo but also is very attractive for use in the bioconversionof D-galactose into D-tagatose in vitro (5, 25) (Fig. 1).

View larger version (21K):
[in this window]
[in a new window]
FIG. 1. Isomerization of an aldose to a ketose by T. maritima AI.

The ketohexose D-tagatose has a sweetness value (92%) equivalentto that of sucrose but is poorly digested (18, 32). This compoundhas been found to be a safe low-calorie sweetener in food productsand is classified as a generally recognized as safe substancein the United States. It has also been classified as generallyrecognized as safe for use in cosmetics and drugs, and it isuseful as a reduced-calorie bulking agent, as an intermediatein the synthesis of other optically active compounds, and asan additive in detergent, cosmetic, and pharmaceutical formulations.Initial quantities of D-tagatose have been produced commerciallyby a patented chemical process since the beginning of 2003 (2).

A number of thermostable xylose isomerases (XIs) (EC 5.3.1.5)have been isolated from extremophiles and have been studiedextensively in connection with the manufacture of high-fructosecorn syrup, because the equilibrium for the isomerization ofglucose to fructose is shifted toward fructose at high temperatures(17, 27, 29).

Our preliminary experiments on the effect of reaction temperatureon the conversion of D-galactose to D-tagatose by Thermotoganeapolitana AI showed that conversion increased as the incubationtemperature was raised (12). For industrial applications ofthe production of D-tagatose from D-galactose, the AI shouldbe thermostable at elevated temperatures (>50°C). Althoughuse of AI for the production of D-tagatose has been reportedrecently, the mesophilic AIs from Lactobacillus gayonii andEscherichia coli have a low affinity for D-galactose (19, 22),and no information is available on thermostable AIs from extremophiles.We describe here biochemical characterization of AI from T.maritima, as well as comparisons with its mesophilic counterpartsand the role of divalent metal ions, such as Mn²⁺ and Co²⁺,in the activity and thermal stability of this enzyme.

MATERIALS AND METHODS

Top

Materials and Methods

Materials.
The reagents used in this study were obtained as follows: restrictionenzymes, Ex-tag DNA polymerase, deoxynucleoside triphosphates,and chemicals for PCR were obtained from Takara Biomedicals;the pGEM-T Easy vector and T4 DNA ligase were obtained fromPromega; the pET-22b(+) expression vector and a His-bind resinkit were obtained from Novagen; genomic-tip and a plasmid miniprepkit were obtained from Qiagen; electrophoresis reagents wereobtained from Bio-Rad; Sephacryl S300 resin was obtained fromPharmacia; and all chemicals used for enzyme assays and characterizationwere obtained from Sigma. Oligonucleotides were synthesizedby Cosmo.

Bacterial strains and culture conditions.
T. maritima DSM 3109 (9) was obtained from Deutsche Sammlungvon Mikroorganismen (DSM), Braunschweig, Germany. T. maritimawas grown in an artificial seawater-based medium (DSM medium343) supplemented with (per liter) 0.5 g of yeast extract, 5g of L-arabinose, 20 g of NaCl, 0.5 g of KH₂PO₄, 0.5 g of Na₂S· 9H₂O, 2 mg of NiCl₂ · 6H₂O, 15 ml of a traceelement solution (DSM medium 141), and 1.0 mg of resazurin.Cultures were grown at 80°C for 48 h in sealed serum bottlesunder N₂ gas. E. coli strains DH5 ${alpha}$ and BL21(DE3) were used asbacterial hosts for recombinant plasmids. The plasmid pGEM-TEasy vector was used as a cloning and sequencing vector, andpET-22b(+) was used for expression.

Cloning, expression, and purification of T. maritima AI.
In a search of the microbial genome sequences in GenBank, wefound a putative araA gene in T. maritima encoding AI. GenomicDNA was isolated from T. maritima and purified by using a genomicDNA extraction kit according to the manufacturer's instructions.The gene encoding AI (araA) was amplified by PCR by using genomicDNA as the template. The PCR mixture (total volume, 50 µl)contained 20 ng of genomic DNA, 10 pmol of primer F-araA-1 (5'-CATATGATAGATCTCAAGCAGTACGAG-3'[an NdeI site is underlined]), 10 pmol of primer R-araA-2 (5'-AAGCTTTCTTTTCAAAAGCCCCCAGTA-3'[a HindIII site is underlined]), 1x PCR buffer, each deoxynucleosidetriphosphate at a concentration of 200 µM, and 2.5 U ofEx-tag DNA polymerase. After an initial denaturation for 4 minat 94°C, the DNA was amplified by 30 cycles consisting of30 s of denaturation at 94°C, 30 s of annealing at 60°C,and 1 min of extension at 72°C, followed by a final extensionstep consisting of 5 min at 72°C. The PCR product was clonedinto the pGEM-T Easy vector and transformed into E. coli DH5 ${alpha}$ competent cells. Transformants containing the pGEM-T Easy vectorharboring the gene encoding T. maritima AI (araA) were selectedon Luria-Bertani medium-ampicillin plates containing 0.01% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-Gal). Plasmid DNA was isolated from the transformants withinserts and digested with NdeI and HindIII. The digested DNAwas purified and ligated into the NdeI and HindIII sites ofpET-22b, yielding pET-TMAI. The expression vector also encodeda C-terminal polyhistidine (six-His) sequence. For expressionof the recombinant enzyme, E. coli BL21 cells transformed withpET-TMAI were grown in Luria-Bertani medium (0.8 liter) containing100 µg of ampicillin per ml at 37°C to an opticaldensity at 600 nm of 0.5 to 0.6. After induction by 1 mM isopropyl-ß-D-thiogalactopyranoside(IPTG), the cells were grown for an additional 5 h and harvestedby centrifugation (10,000 x g, 20 min, 4°C). Bacterial pelletswere stored at -70°C.

The centrifuged cells were suspended in 40 ml of 1x His-bindingbuffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole; pH 7.9) anddisrupted by sonication. The lysate was centrifuged at 14,000x g for 20 min to remove the cell debris, and the supernatantwas heated at 80°C for 20 min. After this the suspensionwas centrifuged at 20,000 x g for 20 min to remove the denaturedE. coli proteins, and the soluble fraction was filtered througha 0.2-µm-pore-size filter. The filtrate was loaded ona His-bind resin column (10 ml) equilibrated with the same buffer.The column was washed with 10 volumes of the same buffer, anda gradient of imidazole (from 5 mM to 1 M) was applied to elutethe recombinant protein. The fractions containing enzyme activitywere pooled and dialyzed against 20 mM Tris-HCl buffer (pH 7.9),and the dialyzed enzyme preparation was stored at 4°C. Proteinconcentrations were determined by the bicinchoninic acid method,with bovine serum albumin as the standard. Enzyme fractionswere analyzed by sodium dodecyl sulfate (SDS)-12% polyacrylamidegel electrophoresis (PAGE) and visualized with Coomassie blue(14).

Enzyme activity assay.
AI (D-galactose isomerase) activity was determined by measuringthe accumulation of L-ribulose (D-tagatose). Unless otherwiseindicated, the standard reaction mixture contained 50 mM HEPESbuffer (pH 7.5 at room temperature), 1 mM CoCl₂, 10 mM MnCl₂,0.2 ml of enzyme preparation at a suitable dilution, 0.1 M L-arabinose(D-galactose), and enough distilled water to bring the finalvolume to 1.25 ml. The reaction mixtures were incubated at 90°Cfor 20 min. The reaction was stopped by cooling on ice. TheL-ribulose (D-tagatose) formed was quantified by the cysteine-sulfuricacid-carbazole method (7), and the absorbance was measured at560 nm. One unit of isomerase activity was defined as the amountof enzyme that produced 1 µmol of product per min underthe assay conditions.

Isoelectric focusing and Western blotting.
Isoelectric focusing was carried out with the PROTEINII ReadyGel precast system (Bio-Rad). The range of the precast gel waspH 3.0 to 10.0. Focusing took place under constant-voltage conditionsin a stepped fashion (15 min at 100 V, 15 min at 200 V, and60 min at 450 V, all at 20°C). For Western blot analysiswith L-arabinose-induced T. maritima cell extracts and polyclonalantibodies raised against purified recombinant T. maritima AI,proteins were electrophoretically transferred to a nitrocellulosemembrane (Bio-Rad), blocked with 3% skim milk in phosphate-bufferedsaline at 4°C overnight, and incubated with polyclonal antibodiesfor 2 h and then with horseradish peroxidase-conjugated mouseanti-mouse immunoglobulin G antibody (Promega) for 60 min atroom temperature. The membranes were washed five times withphosphate-buffered saline containing 0.05% Tween 20 and fivetimes with distilled water and were developed with 3,3',5,5'-tetramethylbenzidene(Promega).

Temperature and pH studies.
The effect of temperature on T. maritima AI activity was measuredby using the standard protocol. To determine the effect of pHon T. maritima AI, the enzyme was incubated under standard assayconditions except that the HEPES buffer was replaced by 50 mMsodium acetate buffer (pH 5 to 6), 50 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonicacid)] buffer (pH 6 to 7.5), 50 mM EPPS [N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonicacid)] buffer (pH 7.5 to 8.5), or 50 mM sodium bicarbonate buffer(pH 9 to 10). All pHs were adjusted at room temperature, andthe ${Delta}$ pK_a/ ${Delta}$ ts (the latter term being the change in temperature)for each buffer were taken into account when the results wereanalyzed.

Effect of metal ions on enzyme activity.
Metal ions were removed from the purified AI by treatment with10 mM EDTA at 60°C for 1 h, followed by overnight dialysisagainst 50 mM HEPES buffer (pH 7.5) at 4°C with severalchanges of buffer. The effects of various metal ions were determinedby adding CoCl₂ · 6H₂O, MnCl₂ · 4H₂O, MgCl₂ ·6H₂O, CaCl₂ · 2H₂O, ZnCl₂ · 6H₂O, CuCl₂ ·2H₂O, FeCl₂ · 6H₂O, or NiCl₂ · 6H₂O at concentrationsof 1 and 10 mM to the dialyzed enzyme and assaying AI activityunder standard conditions without 1 mM CoCl₂ and 10 mM MnCl₂.The dependence of T. maritima AI activity on metal concentrationwas determined by measuring AI activity under standard conditionsafter a 15-min preincubation at 80°C in the presence ofvarious concentrations of metal ions.

Determination of kinetic constants of T. maritima AI.
To determine kinetic parameters, assays were performed in 50mM HEPES (pH 7.5 at room temperature) containing 1 mM CoCl₂,5 mM MnCl₂, and 1 to 800 mM substrate (L-arabinose or D-galactose).Assay mixtures were incubated for 1 and 10 min at 90°C forL-arabinose and D-galactose, respectively, and the reactionswere stopped by cooling on ice.

Analysis of isomerization product.
Thin-layer chromatography of D-galactose and D-tagatose in ethylacetate-isopropanol-water (6/3/1, vol/vol/vol) was performedby using the ascending technique and 0.2-mm silica gel-coatedaluminum sheets (type 60; Merck, Darmstadt, Germany). Each platewas sprayed with 60% concentrated H₂SO₄ and then heated to visualizethe spots (5).

High-performance ionic chromatography (Dionex) was carried outby using a Carbopac PA1 column (Dionex) equipped with a pulsedamperometry electrochemical detector. D-Galactose and D-tagatosewere separated by isocratic elution in a 20 mM NaOH solutionat a flow rate of 0.3 ml/min.

RESULTS AND DISCUSSION

Top

Results and Discussion

Cloning, expression, and purification of T. maritima AI.
The araA gene (1.5 kb) encoding AI was amplified by PCR fromT. maritima genomic DNA and cloned into the pGEM-T Easy cloningvector. For expression in E. coli, as well as to facilitatesubsequent purification, the araA gene was subcloned into thepET-22b(+) expression vector, resulting in pET-TMAI. The genefor AI in pET-TMAI was successfully expressed as a C-terminalhexahistidine-tagged fusion protein in E. coli BL21(DE3) uponinduction with IPTG. To obtain the SDS-PAGE pattern shown inFig. 2A, the E. coli lysates were heated to 80°C to removethe majority of endogenous proteins, while T. maritima AI remainedsoluble and active. A single Ni²⁺ chelate affinity chromatographystep was used to purify the AI to more than 90% purity witha yield of 73%. The apparent M_r of the fusion protein was estimatedto be 57,000 by SDS-PAGE, which is consistent with the M_r (56,658.39)calculated from the presumptive amino acid sequence. The N-terminalsequence of the first 15 residues of the recombinant T. maritimaAI was determined to be MIDLKQYEFWFLVGSQ by the Tufts UniversityAnalytical Core Facility. This is identical to the amino acidsequence deduced from the DNA sequence. In addition, we confirmedby Western blotting with polyclonal antiserum against purifiedrecombinant enzyme that the recombinant AI was identical tothe native AI present in an L-arabinose-induced culture of T.maritima (Fig. 2B). Thus, we successfully overexpressed andpurified recombinant T. maritima AI.

View larger version (40K):
[in this window]
[in a new window]
FIG. 2. SDS-PAGE, Western blotting, and isoelectric focusing analyses of the recombinant T. maritima AI. (A) Purified recombinant T. maritima AI was analyzed by SDS-12.5% PAGE. Lane 1, molecular weight markers; lane 2, crude extract of E. coli BL21(pET-TMAI); lane 3, crude extract after heat treatment; lane 4, Ni²⁺ affinity pool (5 µg). (B) Western blotting of recombinant and native AIs from E. coli and T. maritima with mouse antiserum raised against recombinant AI. Lane 1, crude extract of uninduced E. coli BL21(pET-TMAI); lane 2, crude extract of induced E. coli BL21(pET-TMAI); lane 3, L-arabinose-induced crude extract of wild-type T. maritima. (C) Purified recombinant AI (10 µg) analyzed by isoelectric focusing (pH 3.0 to 10.0).

Biochemical characterization of T. maritima AI.
As described above, the theoretical M_r of T. maritima AI was56,658.39, and SDS-PAGE confirmed that the apparent M_r was 57,000.However, gel filtration chromatography on Sephacryl S300 yieldedan estimated M_r of 230,000 (Fig. 3), suggesting a homotetramericstructure, compared with the homohexameric structure of E. coliAI (molecular mass, 362 kDa) (22, 30). The experimentally determinedisoelectric point of the recombinant T. maritima AI was pH 5.7(Fig. 2C).

View larger version (19K):
[in this window]
[in a new window]
FIG. 3. Time course of tagatose production during T. maritima AI-catalyzed isomerization of D-galactose. The reaction mixtures (1 ml) contained 50 mM HEPES buffer (pH 7.5), 10 mM D-galactose, 1 mM Co²⁺, 5 mM Mn²⁺, and 5 mg of T. maritima AI. Aliquots were withdrawn after various periods of incubation at 70°C ( ${square}$ ), 80°C (•), and 90°C ( ${circ}$ ).

The temperature dependence of the recombinant enzyme was determinedin the presence and absence of divalent metal ions (1 mM Co²⁺and 5 mM Mn²⁺) after 20 min of incubation at various temperatures(Table 1). In the presence of metal ions, the apparent optimumtemperature for the recombinant enzyme was 90°C, whereasin the absence of metal ions the apparent optimum temperaturewas 85°C. The slight decrease in the apparent optimum temperaturemay reflect an effect of metal ions on enzyme conformation.This phenomenon is discussed further below. The apparent optimumpH at 90°C in the presence of 1 mM Co²⁺ and 5 mM Mn²⁺ was7.5 (Table 1).

View this table:
[in this window]
[in a new window]
TABLE 1. Biochemical properties of T. maritima and other microbial AIs

AI is known to convert D-galactose into D-tagatose and to convertL-arabinose into L-ribulose (5, 10). However, it has been notedthat D-galactose, D-fucose, and L-arabinose are extremely poorsubstrates for AIs from mesophilic microorganisms (3, 11, 19,22). In particular, the AIs from E. coli and L. gayonii havenegligible activity for converting D-galactose into D-tagatose(19, 22). Figure 3 shows the time course of D-tagatose productionfrom D-galactose by the purified T. maritima AI at a range oftemperatures. Aliquots of the reaction mixture were withdrawnperiodically and analyzed by thin-layer chromatography and high-performanceionic chromatography. The ratio of conversion of D-galactoseto tagatose at 80°C was higher than that at 70°C. Thisis consistent with the previous results for T. neapolitana AIthat showed that the ratio of conversion of D-galactose to D-tagatosedepends on the reaction temperature (12). Maximum yields of56 and 50% conversion of D-galactose to D-tagatose could beobtained after 6 h of incubation at 80 and 70°C, respectively.However, at 90°C the enzyme became inactivated before theequilibrium was reached. L-Arabinose, a major substrate forAI, was converted to L-ribulose with a 70% yield in 6 h at 80°Cunder the same conditions (data not shown). Even though we couldnot conclude that the affinity of T. maritima AI for D-galactoseis unusually high, it seems clear that T. maritima AI catalyzesthe conversion of D-galactose into D-tagatose very efficientlycompared to mesophilic AIs (12) and that, at equilibrium, theratio of D-galactose to D-tagatose is dependent on the reactiontemperature.

Comparison of the protein sequences.
The amino acid sequence of T. maritima AI was compared withthose of other mesophilic AIs. Table 2 shows the percentagesof identity and similarity for reported AI sequences. The T.maritima AI exhibits high levels of sequence similarity to thermolabileAIs from E. coli (72%), Salmonella enterica serovar Typhimurium(72%), Bacillus halodurans (73%), and Bacillus subtilis (71%),as well as thermostable AIs from T. neapolitina (98%) and Geobacillusstearothermophilus (76%). Interestingly, the AIs have very lowsequence homology (<10%) with other sugar isomerases (e.g.,XI, fucose isomerase, etc.) (data not shown).

View this table:
[in this window]
[in a new window]
TABLE 2. Comparison of amino acid sequences of AIs from various microorganisms^a

As shown in Fig. 4, an amino acid sequence alignment of severalAIs with the enzyme from T. maritima revealed that these enzymescontain several highly conserved domains. Secondary structureanalysis demonstrated that the secondary structure elementsare also highly conserved in these microbial AIs (Fig. 4). Itis difficult to identify differences in secondary structurebetween T. maritima AI and other microbial AIs. However, AIsfrom mesophiles and thermophiles differ greatly in stability(Table 1), but as no AI crystal structure is available, it isnot yet possible to account for these differences. Nonetheless,we were able to pinpoint a few characteristic features by comparingthe amino acid compositions of T. maritima AI and other microbialAIs (Table 3). The pI of T. maritima AI is higher than thatof mesophilic AIs due to a higher content of basic amino acidsand a lower content of acidic amino acids. Another characteristicof T. maritima AI is that its hydrophobic amino acid contentis higher than that of mesophilic AIs. When hydrophobicity wasestimated from the grand average of hydropathicity index (http://www.expasy.org/tools/protparam.html)(13), the value for T. maritima AI was greater than that formesophilic AIs, indicating that T. maritima AI is much morehydrophobic than other mesophilic AIs. This result is consistentwith the fact that hydrophobic interactions are important forprotein thermostability. Moreover, the content of charged aminoacids in T. maritima and T. neapolitana AIs is higher than thatin mesophilic enzymes. This suggests that the higher contentof charged amino acids in thermostable AIs may be an adaptationto enhance the quaternary structure of the enzymes by generatinga network of intersubunit ion pairs or salt bridges.

View larger version (101K):
[in this window]
[in a new window]
FIG. 4. Alignment of the amino acid sequences of T. maritima and other microbial AIs. The residues of T. maritima AI involved in ${alpha}$ -helices or ß-strands are indicated by H and E, respectively. Secondary structure prediction was carried out by using the PROFsec (B. Rost; http://maple.bioc.columbia.edu/predictprotein) and Gor4 (8) programs. TMAI, T. maritima AI (National Center for Biotechnology Information protein database accession no. AE001709); TNAI, T. neapolitana AI (AY028379); GSAI, G. stearothermophilus AI (AF160811); ECAI, E. coli AI (M15263); BSAI, B. subtilis AI (1769994); STAI, S. enterica serovar Typhimurium AI (M11047) (16); MSAI, Mycobacterium smegmatis AI (7242883).

View this table:
[in this window]
[in a new window]
TABLE 3. Properties of T. maritima AI and other microbial AIs determined from a comparison of the deduced amino acid sequences

Effect of metal ions on enzyme activity and stability.
To investigate the effect of divalent metal ions on AI activity,the purified enzyme was dialyzed against 5,000 volumes of 50mM HEPES buffer (pH 7.5) containing 10 mM EDTA. No activitywas measurable in the absence of divalent metal ions. However,activity was recovered when Mn²⁺ or Co²⁺ was added. Thus, likeall other L- and D-AIs previously reported (3, 12, 19, 22, 31),T. maritima AI has a requirement for Mn²⁺ or Co²⁺. As shownin Fig. 5A, about 1 mM Co²⁺ restored activity, whereas 5 mMMn²⁺ was needed for full activity. Compared to Mn²⁺ and Co²⁺,other metal ions, such as Mg²⁺, Ca²⁺, Fe²⁺, and Ni²⁺, were pooractivators (data not shown). It is evident that Mn²⁺ and Co²⁺are effective activators. Although the mesophilic AI from L.gayonii was slightly activated by Co²⁺, Mn²⁺ is the only activatingmetal ion for E. coli AI. We performed kinetic experiments onthe effect of Mn²⁺ and Co²⁺ on T. maritima AI. Double-reciprocalplots of velocity versus substrate concentration and velocityversus metal ion concentration gave a family of lines in whichthe slope and intercept on the vertical axis varied with theCo²⁺ concentration as well as the Mn²⁺ concentration (Fig. 5B and C).The results of these kinetic studies reflect the factthat not only Mn²⁺ but also Co²⁺ is able to activate T. maritimaAI. However, it was found that there is a decrease in velocityat high metal concentrations, as observed for substrate inhibition.This was manifested by an upward curvature at low values of1/[Co²⁺], where an upward curvature is observed at cobalt concentrationsabove 5 mM (Fig. 5C). This phenomenon is known as substrateinhibition and is usually interpreted in terms of the existenceof two types of substrate-binding sites in the enzyme. At highmetal concentrations, a low-affinity type of site becomes occupied,and this is presumed to inhibit the catalytic reaction takingplace at the high-affinity type of site. For a fuller investigationof this phenomenon, the three-dimensional structure of the enzymecomplex with divalent metal ions should be investigated.

View larger version (16K):
[in this window]
[in a new window]
FIG. 5. Effect of concentration of divalent metal ions on the activity of purified T. maritima AI. (Top) EDTA-pretreated T. maritima AI (0.5 mg/ml) activity was measured at various Mn²⁺ ( ${circ}$ ) and Co²⁺ (•) concentrations in 50 mM HEPES buffer (pH 7.5) containing 40 mM L-arabinose. (Middle and bottom) The purified enzyme (0.5 mg/ml) was treated with 10 mM EDTA and then dialyzed against 50 mM HEPES buffer (pH 7.5) overnight at 4°C. The EDTA-treated and dialyzed enzyme was incubated at 90°C for 1 min in the presence of 0.1 mM Mn²⁺ ( ${blacktriangleup}$ ), 1 mM Mn²⁺ (•), or 10 mM Mn²⁺ ( ${blacksquare}$ ) (middle) or in the presence of 2.5 mM Co²⁺ (•), 5 mM Co²⁺ ( ${circ}$ ), 10 mM Co²⁺ ( ${blacktriangleup}$ ), 20 mM Co²⁺ ( ${triangleup}$ ), and 40 mM Co²⁺ ( ${blacksquare}$ ) (bottom).

Interestingly, T. maritima AI also required Mn²⁺ or Co²⁺ forthermostability (Fig. 6). The apoenzyme proved to be very unstableat 90°C in the absence of metal ions. Addition of 5 mM Mn²⁺or 1 mM Co²⁺ significantly increased the thermostability. Indeed,incubation in the presence of Mn²⁺ or Co²⁺ at 80°C for 240min resulted in little loss of activity.

View larger version (18K):
[in this window]
[in a new window]
FIG. 6. Time course of irreversible thermal inactivation of purified T. maritima AI. After various periods of incubation at 80°C (top) and 90°C (bottom) in the absence of divalent metal ions ( ${circ}$ ) or in the presence of 1 mM Co²⁺ (•) or 5 mM Mn²⁺ ( ${square}$ ), aliquots were withdrawn, and their residual activities were measured under the standard conditions.

It has been noted that all of the simple sugar isomerases, suchas XI and fucose isomerase, are metalloproteins, and it hasbeen suggested that the metal plays the same role in these enzymesthat phosphate plays in the phosphosugar isomerases (1). Nuclearmagnetic resonance experiments with L-arabinose showed thatAI isomerizes L-arabinose to L-ribulose by exchanging the protonat carbon 2 of the substrate (1). The presence of Mn²⁺ in E.coli B/r produces an enzyme with greater intrinsic activityand heat stability (23), and divalent metal ions have also beenfound to be required for the thermal stability of other thermostableenzymes. For example, the XIs of T. maritima, T. neapolitana,and Thermoanaerobacterium strain JW/SL-YS 489 require metalions, such as Mn²⁺ and Co²⁺, for heat stability, as well ascatalytic activity (17, 29). Thermostable enzymes, such as aminoacylasesand carboxypeptidases, are known to contain Zn²⁺ ions that maintainor stabilize the active conformation (26, 28). Ca²⁺ also enhancedthe thermostability of an extracellular amylase from Thermococcusprofundus DT5432 (6).

In order to exploit quantitative data for the fraction of foldedand unfolded protein as a function of temperature, the changein ellipticity at 225 nm was monitored. The effects of Mn²⁺and Co²⁺ on the thermal transition of T. maritima AI were determined,as shown in Fig. 7. In the absence of metal ions, the apoenzymebegan to denature at 80°C, and about 50% of the total enzymewas denatured at 88°C. On the other hand, in the presenceof Mn²⁺ and Co²⁺, over 50% of the enzyme remained in the nativestate at 94°C. Divalent metal ions appear, therefore, toincrease the thermostability of the enzyme by at least 5 to6°C. Hence, we propose that divalent metal ions, such asMn²⁺ and Co²⁺, are key stabilizing factors for the tetramericstructure of T. maritima AI.

View larger version (18K):
[in this window]
[in a new window]
FIG. 7. Thermal transitions of T. maritima AI in the presence of 5 mM Mn²⁺ ( ${square}$ ) or 1 mM Co²⁺ (•) or in the absence of divalent metal ions ( ${circ}$ ). Each purified enzyme sample (0.5 mg/ml) in 20 mM HEPES buffer (pH 7.5) was scanned at temperatures from 30 to 110°C. The temperature was increased at a constant rate of 1°C/min. Protein unfolding was monitored by determining the temperature-induced changes in ${alpha}$ -helical ellipticity at 225 nm. The fraction of unfolded protein was calculated as described previously (21).

Determination of kinetic parameters of T. maritima AI.
The kinetic parameters were determined for L-arabinose and D-galactoseas substrates from Lineweaver-Burk plots. The K_m for L-arabinosewas 31 mM, and the V_max was 41.3 U/mg, whereas for D-galactosethe K_m and V_max were 60 mM and 8.9 U/mg, respectively. The catalyticefficiency (k_cat/K_m) for L-arabinose (74.8 mM^-1 · min^-1)was approximately nine times higher than that for D-galactose(8.5 mM^-1 · min^-1). Although a direct comparison is difficultbecause of the different reaction temperatures, T. maritimaAI appears to have a lower K_m and a higher efficiency than T.neapolitana AI (12). However, with D-galactose and L-arabinose,the V_max of T. maritima AI was lower than that of T. neapolitanaAI (the V_max values were 41.3 and 8.9 U/mg at 90°C, comparedto 119 and 14.3 U/mg for the T. neapolitana AI at 85°C).

The biochemical properties of T. maritima AI are compared withthose of mesophilic AIs in Table 1. The differences betweenthese microbial AIs are very interesting in view of the similaritiesof the primary and secondary structures of the molecules. Sinceno three-dimensional AI structure is available, these differencescannot be explained yet. For industrial applications, the catalyticefficiency for D-galactose needs to be increased for the enzymesto be useful in the commercial production of tagatose as a low-caloriebulk sweetener. The productivity of the enzymes can be enhancedby addition of metal ions, such as Co²⁺, Mn²⁺, or Mg²⁺, etc.,and the nature of the selected ion and its optimum concentrationare different for different enzymes. However, certain ions,such as Co²⁺, cannot be used in nutritional applications. Tosatisfy these requirements, it would be desirable to modifythe enzymes by directed evolution. In this connection an X-raycrystallographic study of AI would be desirable to facilitaterational structure-based mutagenesis, as has been done for similarXIs. Since AIs from mesophiles and (hyper)thermophiles differonly in a small number of amino acids, the possible use of systematicor random mutagenesis to correlate increases in stability withspecific amino acid substitutions seems most promising. T. maritimaAI may be a good model for analysis of metal-mediated thermostabilizationand for industrial application in the production of D-tagatoseas a novel sweetener.

ACKNOWLEDGMENTS

This work was supported by grant 2001-2-0109 from the KoreaScience and Engineering Foundation and by grant AIC-08-02 fromMinistry of Commerce, Industry and Energy, Korea.

We gratefully acknowledge Julian Gross for helpful discussionsand editing the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biotechnology and Bioproducts Research Center, College of Engineering, Yonsei University, Seodaemun-Gu, Shinchon-Dong 134, Seoul 120-749, Korea. Phone: 82-2-2123-2883. Fax: 82-2-312-6821. E-mail: yrpyun{at}yonsei.ac.kr.

REFERENCES

Top