Vol. 174, No. 16JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5400-54050021-9193/92/165400-06$02.00/0Copyright ) 1992, American Society for Microbiology

Structure of the Gene Encoding Cyclomaltodextrinase fromClostridium thermohydrosulfuricum 39E and Characterization

of the Enzyme Purified from Escherichia coliSERGEY M. PODKOVYROV1 AND J. GREGORY ZEIKUS12*

Department ofBiochemistry' and Department ofMicrobiology and Public Health, 1'2Michigan State University, East Lansing, Michigan 48824

Received 6 April 1992/Accepted 15 June 1992

Clostridium thermohydrosulfuricum 39E, a gram-positive thermophilic anaerobic bacterium, produced acyclodextrin (CD)-degrading enzyme, cyclodextrinase (CDase) (EC 3.2.1.54). The enzyme was purified tohomogeneity from Escherichia coli cells carrying a recombinant multicopy plasmid that contained the geneencoding for thermophilic CDase. The purified enzyme was a monomer with an Mr of 66,000 ± 2,000. Itshowed the highest activity at pH 5.9 and 65°C. The enzyme hydrolyzed a-, j3-, and 'y-CD and linearmaltooligosaccharides to yield maltose and glucose. The Km values for a-, 0-, and fy-CD were 2.5, 2.1, and 1.3mM, respectively. The rates of hydrolysis for polysaccharides (starch, amylose, amylopectin, and pullulan)were less than 5% of the rate of hydrolysis for a-CD. The entire nucleotide sequence of the CDase gene wasdetermined. The deduced amino acid sequence of CDase, consisting of 574 amino acids, showed somesimilarities with those of various amylolytic enzymes.

Cyclodextrins (CDs) are cyclic oligosaccharides consist-ing of a-1,4-linked 6-, 7-, or 8-glucopyranose units usuallyreferred to as a-, 1-, or -y-CDs, respectively. One of theremarkable properties of the CDs is their resistance tohydrolysis by the common starch-splitting enzymes. Amongthe bacterial amylases, only those from Bacillus subtilis (27),Pseudomonas sp. (14), and Flavobacterium sp. (2) catalyzethe hydrolysis of CDs; for the first of these enzymes, CDsare poor substrates, while the latter two enzymes degradeCDs faster than starch. Until now, only four strains, B.macerans (7), B. coagulans (16), B. sphaericus (29), and aBacillus sp. (43), have been known to produce cyclomalto-dextrinase (EC 3.2.1.54, cyclomaltodextrin hydrolase, decy-cling) (CDase), an enzyme which effectively hydrolyzes CDsand linear maltodextrins. In contrast to the extensive infor-mation concerning amylolytic enzymes, studies on CDaseshave been confined to these four enzymes. To our bestknowledge, nothing to date has been reported about the genestructure of CDases.

In the course of our studies on thermophilic bacteria, werecently characterized a thermophilic CDase from Clostnid-ium thermohydrosulfuricum 39E (33). This enzyme waspartially purified (about 205-fold), and many of its propertieswere determined. However, because the CDase was notpurified to homogeneity, important molecular and kineticproperties of the enzyme remain unknown. In the presentstudy, we report on the purification and detailed character-ization of C thermohydrosulfuricum 39E CDase from Esch-erichia coli harboring the recombinant multicopy plasmidpPG22. Initially, this plasmid was selected from a genomiclibrary of C. thennohydrosulfuricum 39E for its ability toconfer pullulanase activity in E. coli cells (18). However,after the "pullulanase" from the recombinant E. coli strainwas purified, kinetic studies revealed that hydrolysis rateswith CDs were much higher than those with pullulan. Thus,this cloned gene is more properly designated a gene encoding

* Corresponding author.

for CDase. Here, we present the sequence of this C. ther-mohydrosulfuricum 39E CDase gene and analysis of thededuced amino acid sequence.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. E. coilDH5a [F- recAl A(lacZYA-argF)U169 hsdR17 (rK- MK+)thi-1 X- gyrA96 supE44 endAl relAI +80d lacZAM15] wasobtained from Bethesda Research Laboratories (Gaithers-burg, Md.). Plasmid pPG22 was constructed previously (18).The cells of DH5a(pPG22) were grown at 37°C in Luria-Bertani media (22) containing 50 ,ug of ampicillin per ml.DNA sequencing and analysis. Plasmid DNA for sequenc-

ing was purified by CsCl-ethidium bromide density gradientcentrifugation (22). Templates were prepared by using thealkaline denaturation procedure (44). The DNA sequencehas been determined on both strands by the chain termina-tion method (34) with the Sequenase kit (U.S. Biochemical,Cleveland, Ohio) and 35S-ATP (Du Pont Co., Wilmington,Del.). Single-stranded oligonucleotide primers were de-signed from the previously sequenced region and weresynthesized in the Macromolecular Structure Facility, De-partment of Biochemistry, Michigan State University. Se-quence analysis was performed with University of Wiscon-sin Genetics Computer Group Sequence Analysis PackageVersion 6.2 (8).Enzyme assay. CDase activity was assayed in the reaction

mixture that contained 1% 1-CD in 50 mM sodium acetatebuffer (pH 6.0) and appropriately diluted enzyme. Afterincubation at 65°C for 30 min, the reducing sugar wasmeasured by the dinitrosalicylic acid method (3). One unit ofactivity was defined as the amount of enzyme that released 1,umol of reducing sugar per min with glucose as the standard.

Protein determination. Protein was determined by usingthe method of Bradford (5) with bovine serum albumin as thestandard.Enzyme purification. All of the procedures were carried

out at 4°C. E. coli DH5a(pPG22) cells were grown to late log

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TABLE 1. Summary of CDase purification

Total Total Spat PrfcioStep protein activity (U/mg) (fold)(mg) (U) (Um) fod

Cell-free extract 510 2,550 5 1DEAE-Sepharose CL-6B 13 611 47 9.4

(anion exchange)G3000SW TSK-GEL (gel 4.5 279 62 12.4

filtration)

phase, centrifuged, and washed twice in 50 mM acetatebuffer (pH 6.0). Cells (15 g) were resuspended in 100 ml ofthe same buffer, disrupted by 1 passage through a Frenchpressure cell (American Instrument Co., Silver Spring, Md.)at 124 MPa, and centrifuged at 15,000 x g for 20 min. Thesupernatant was concentrated by ultrafiltration (YM 30membrane; Amicon Co., Danvers, Mass.) and applied to aDEAE-Sepharose CL-6B column (2.6 by 38 cm) preequili-brated with 50 mM acetate buffer (pH 6.0). The column waswashed extensively with the same buffer, and the adsorbedenzyme was eluted with a linear gradient of sodium chloridefrom 0 to 0.5 M in the same buffer. The active fractions werepooled, concentrated by ultrafiltration, and loaded on aG3000SW TSK-GEL column (TosoHaas, Philadelphia, Pa.)by using a Waters 650E FPLC system (Waters, Milford,Mass.). The fractions containing CDase were pooled andconcentrated with Centricon-30 microconcentrators (Ami-con).M, determination. The molecular weight of the denatur-

ated protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21) withlow-molecular-weight standards (Bio-Rad Laboratories,Richmond, Calif.). The Mr of the native enzyme was deter-mined by gel filtration on a G3000SW TSK-GEL column in aWaters FPLC system, with apoferretin (443,000), P-amylase(200,000), alcohol dehydrogenase (150,000), and bovine se-rum albumin (66,000) as Mr standards.

N-terminal amino acid sequence determination. The proteinpattern was prepared by washing the purified CDase withdouble-distilled water five times with a Centricon-30 micro-concentrator (Amicon) to remove contaminating salts andbuffer from the solution. The N-terminal amino acid se-quence was identified by using a protein sequencer model477A (Applied Biosystems, Foster City, Calif.) in the Mac-romolecular Structure Facility, Department of Biochemis-try, Michigan State University.

Thin-layer chromatography. CDase hydrolysates were an-alyzed by thin-layer chromatography on high-performancesilica gel HP-K plates (Whatman International Ltd., Maid-stone, United Kingdom) with the mobile phase consisting ofbutanol-ethanol-water (3:2:2). The spots were visualizedafter plates were treated with a mixture of 0.2% orcinol inmethanol and 20% H2SO4 in methanol (1:1, vol/vol) and thenincubated at 150°C for 2 min.

Nucleotide sequence accession number. The sequence datapublished in this paper have been assigned the GenBankaccession number M88602.

RESULTS

Purification and properties of CDase. The results of thepurification procedure are summarized in Table 1. Thethermophilic CDase was purified 12-fold from E. coli with an11% yield. The purified enzyme migrated as a single protein

100

6-

80

260

<, 40r

20

4 6 10 12

pH

100

-

0-

IL)

C)._._

a:

80

60

40

20 _45 50 55 60 65 70 75 80

Temperature (°C)

FIG. 1. Effects of pH (a) and temperature (b) on the activity ofCDase. The effect of pH on CDase activity was determined by usingthe assay described in Materials and Methods at 65°C, and the effectof temperature on CDase activity was determined at pH 6.0.

band with a molecular mass of approximately 65 kDa inSDS-PAGE. Gel filtration of the native CDase resulted in theestimation of the molecular mass as 67 kDa. Taken together,these data show that CDase is a monomer with a molecularmass of approximately 66 kDa. The N-terminal amino acidsequence determined for the CDase was Met-Ile-Lys-Gly-Ala.The highest enzyme activity was observed at pH 5.9 and

65°C (Fig. 1). The enzyme activities at different temperatureswere determined by the measurement of the reducing sugarformed during incubation of P-CD and enzyme in acetatebuffer (pH 6.0) for 30 min. Incubation of the CDase at 65°Cfor 1 h led to a 15% loss of enzyme activity. The influence ofselected metal ions and chemicals on CDase activity wasinvestigated (Table 2). The enzyme was inhibited by Ag+,Hg2+, Cu2+, Zn2+, p-chloromercuribenzoate, and N-bromo-succinimide, and no activators were found.A number of a-glucans were incubated with the purified

CDase in order to ascertain the substrate specificity. Asshown in Table 3, CDase preferentially hydrolyzed a- andP-CD. The enzyme had a high specificity for maltooligosac-charides, especially for maltoheptaose, but pullulan, amy-lopectin, amylose, and starch were hydrolyzed at rates muchlower than that of CDs.The Michaelis constants for CDs were obtained from

Lineweaver-Burk plots of specific activities at various sub-strate concentrations. The substrates were dissolved in 50mM acetate buffer (pH 6.0). For each assay, 200 ,il of

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TABLE 2. Effect of metal ions and inhibitors on CDase fromC thermohydrosulfuricum 39E'

Metal or inhibitor Activity(mM) (%)

None. 100Ca2+ (5)..................................................... 99K+ (5) ............................................ 98Ni2+ (5)......................................................... 91C02+ (5).................................................................... 91

Mg2+ (5)............................. 89Mn2+ (5)............................................. 68Cu2+ (1) ............................................ 2Ag+ (1) .......... .................................. 2Hg2+ (1) ............................................ 1Zn2+ (1)..................................................... 1EDTA (10) ............ ................................ 82SDS (0.1%) ............. ............................... 70N-Bromosuccinimide (0.03)........................................... 9p-Chloromercuribenzoate (0.1)....................................... 3

a Enzyme activity was measured as described in Materials and Methods,and the addition of the substrate was made after incubation of the enzymewith metal or inhibitor for 5 min.

substrate and 20 ,ug of purified enzyme (1 ,ul) were incubatedfor 10 min at 65°C. The reducing sugar formed from thesubstrate was measured as described in Materials and Meth-ods. The Km values for a-, 1-, and y-CD were found to be2.5, 2.1, and 1.3 mM, and the maximum initial velocitieswere 71, 62, and 21 U/mg, respectively.The products from the hydrolysis of various substrates by

CDase were examined by thin-layer chromatography. Timecourse analysis of CD hydrolysis indicated that at an earlystage of the reaction the three cyclic dextrins were hydro-lyzed to the corresponding linear dextrins, which weresubsequently degraded to smaller dextrins. At the final stageof digestion, only maltose and glucose were detected, withmaltose as the main product. Five linear maltodextrinsranging from maltotriose to maltoheptaose were hydrolyzedto maltose and glucose; however, isomaltotriose and maltosewere not split. Pullulan, amylose, amylopectin, and starchwere degraded slowly, releasing very small amounts ofmaltose and glucose.

TABLE 3. Hydrolysis of various substrates by CDase fromC. thennohydroswulficum 39E4

Substrate Relative rate (%)of hydrolysis

a-CD ...................................... 100P-CD ...................................... 87y-CD...................................... 29Maltose...................................... 0Maltotriose ...................................... 8Maltotetraose...................................... 23Maltopentaose...................................... 32Maltohexaose...................................... 38Maltoheptaose...................................... 42Amylose ...................................... 2Amylopectin ...................................... 3Pullulan ......................................5Soluble starch .................... .................. 3Glycogen...................................... <0.1

a Substrates were prepared in 50 mM acetate buffer (pH 6.0) at finalconcentrations of 2 mM for CDs and oligosaccharides and 0.2% for macro-molecular polysaccharides. A suitable quantity of enzyme was added toproduce a linear increase in reducing sugar during the first 20 min of thereaction.

Nucleotide sequence of the CDase gene. Plasmid pPG22,which was used for the CDase gene sequencing, consisted ofa pUC19 vector (42) and a 3.8-kb DNA fragment from C.thermohydrosulfuricum 39E. The primary structure of the 5'region of the cloned gene was determined previously (18). Inthe present study, the first primer for DNA sequencing wassynthesized according to this known structure. The resultingnucleotide sequence of 2,453 bp is given in Fig. 2. A searchof the sequence for possible protein-encoding regions re-vealed only one large open reading frame (ORF). It encodeda protein consisting of 574 amino acids with a predictedmolecular weight of 68,028, which is comparable to theestimated molecular weight of the purified CDase. Thededuced amino acid sequence from this ORF matched theN-terminal amino acid sequence of the CDase determined byprotein sequencing. It was therefore concluded that theidentified ORF was the structural gene coding for the Cthennohydrosulfuricum 39E CDase.The structural gene was preceded by a sequence similar to

the bacterial -35 and -10 consensus promoter sequences(17). The distance between the -35 and -10 regions was 17bp. The presence of TG at positions -16 and -15, which isparticularly striking for the promoters of gram-positive bac-teria (10), was also found. A region corresponding to theShine-Dalgarno sequence (17) was identified 7 nucleotidesupstream from the translational start codon. Two stopcodons followed close to the stop codon of the CDasestructural gene in the same reading frame, ensuring thetermination of translation. A palindromic sequence whichmay be responsible for p-independent termination of tran-scription (31) or mRNA stability (35) was observed in the 3'flanking region of the ORF.The G+C content of the ORF was slightly lower (33%)

than that of the chromosomal DNA (37%) (41). The G+Ccontent of the third codon position was also low (31%) andreflected a bias for selection of codons ending in A or U. Thecodon usage of the CDase gene appeared to be closelyrelated to that observed for Clostridium and Bacillus genes(11, 25) and was not optimal for the E. coli translationmachinery (9).Homology of CDase with functionally related enzymes. The

amino acid sequence of the CDase was compared with theprimary structures of ot-amylases from B. amyloliquefaciens(38), B. stearothermophilus (37), B. licheniformis (40), andAspergillus oryzae (Taka-amylase A) (39), a-amylase-pullu-lanase from C. thermohydrosulfuricum (26), pullulanasefrom Kiebsiella aerogenes (15), neopullulanase from B.stearothermophilus (19), isoamylase from Pseudomonasamyloderamosa (1), and cyclodextrin glucanotransferase(CGTase) from Bacillus sp. (12) (Fig. 3). Three highlyconserved regions were found. On the whole, CDase mostclosely resembled neopullulanase from B. stearothermophi-lus and a-amylase-pullulanase from C. thennohydrosulfuri-cum. There were two additional sequence similarities be-tween these enzymes, one before the first region and theother between the second and the third regions (Fig. 3).Alignment of the CDase sequence with the amino acidsequence of glucoamylase from Aspergillus niger (4) did notyield regions of extensive homology.

DISCUSSION

To the our best knowledge, this article contains the firstreported sequence of a gene encoding for a CDase. TheCDase investigated in this study is an enzyme from thethermophilic bacterium C. thernohydrosulfuricum 39E. It is

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CYCLODEXTRINASE GENE FROM C. THERMOHYDROSULFURICUM 5403

ACT TGC ACC AAT TCC ACA ACA AAA TCA AAG TGA AGC TTC AAC AAT

TTC GGA AGA CAA AAC AAA TCT TCC ACA GGA AAG CAA TGT TTC TCA

ACC TAT AGT AAA GACGTC OAT TGA AAA AGT AGC AGA GAC TTC TAA

AAA AAT AGA ATA TAA AGT TGT TCC CTT TGG AGA AAG TTA TAA AGT

AGA AAT AAG CGT ACC TGG CGA AAA ATG TTG OTT TAO TGT AAA AGT

AGA TGG AAA TOT CGT TTA TOA AGG CCT TAT GAC AAA GGA TAT GTC

TAA AAT TTT TGA TGT AAA AGA TAG TAT TAC TAT ATT CAT CC0 ATA-35

TCC 0CC AOC TGT TAA AAT AAC TGT AGA C00 TGA GGA GCT ACC AAC

-10

AGT ACA TAC ACC ATC ACC GGT TAC TAT AAA TAT TCA TCC ATA GAA

AGG AGA AAA AAG ATG ATA AAA GAA GCT ATA TTC CAT AAA ACC OAT

SD n I K Z A I H K S D

CTG CCT TAC CCA TAT CCA CTG AAT GAA AAT CAA TTA AAC ATA GTAP Y A Y P L N Z N Q L K I

V

CTA AGO ACT OCT GTA TTT OAT CTA GAT AGG GTG TAT OTT TTA TACL R T A V D v D R v r v L Y

AAA GAC AGA TAC GAT TGG CTG GGA AAA TTC AAA ATA AAA CCA ATGK D R Y D W L K P K I K P M

GTA TTA ACT CAT ACC AAC GACG TTA TTT OAT TAT TAT GAG ACT ACTV L T H T N e L P D Y Y Z T T

TTA GAG CTT AAT AAA AAA TTT GTG TAT TTT TTC TAT TTGGTA TCT

L N K K P V Y P P Y L V S

GAT GCG GGC GAG AAG TTA TAT TAT ACA GAAGCA GGA TTT TAC AAA

D G e K L Y Y T e A G P Y K

AAA AGG CCA GAA AAT CAT TTT TGG GGA TTT TTC CAT TAC CCG TAT

K R p e N H P W G P H Y P y

ATA GGA GAA AAG GAT GTC TTT TTT GCT CCA GAG TGO ACA AGC OAT

I G e K D V P P A P e w T S D

TGC ATG GTG TAT CAG ATA TTT CCT GAA AGG TTT AAT AAC GGT OAT

C M V Y Q I P P e R P N N D

AAA TCA AAT GAC CCT GAA AAT OTA AAG CCC TGG GGT OAA AAA CCTK S N D p e N v K P w e x P

ACT GCA GAT TCT TTC TTT GGA GGA OAT TTG CAA GGA ATA ATA GATT A D S F F G D L Q I I D

AAA ATA OAT TAT TTA A-AG AT TTG GOT ATA AAT GCC ATT TAT TTA

K I D Y L K D L G I N A I Y L

ACC CCC ATA TTT TTA TCO CAT TCC ACC CAC AAA TAT OAT ACT ACT

T P I L S H S T K Y D T T

OAT TAT TAT ACA ATT GAC CCC CAC TTT GGT GAT ACG CAA AAA CC0D Y Y T I D P H D T Q K A

AGA GAA CTT GTO CAA AAA TOT CAT GAC AAT 0GC ATA AAA OTT ATA

R e L V Q K C D N I K v I

TTT GAT GCC GTT TTT AAT CAC TOC GGA TAT GAC TTT TTT GCA TTT

P D A V P N C G Y D F P A P

CAG GAT GTT ATT AAA AAT GOT AAA AAA TCA AAA TAC TOG OAT TGGQ D V I K N K K S K Y w D

TTT AAT ATA TAC OAA TGG CCA ATT AAA ACT CAT GGA AAA CCT TCCP N I Y e w P I K T G K P S

TAT OAA GCT TTT GCA CAT ACT GTA TGG AGA ATG CCA AAA CTT ATGY e A F A D T v w R M P K L M

ACA AAA AAT CCA GAA GTA CAG AAG TAT TTA TTG GAA OTA 0C0 CAAT K N P e v Q K Y L L r V A 8.,

TAT TOG ATT AAA CAA GTG GAC ATA CAC 0C0 TOO AGO CTG CAT GTTY I K E v D I D G R L D V

OCT AAT GAO ATA GAC CAC CAT TTT TGO AGA AAO TTT AGA CAA OTT

A N e I D H H P w R K R I v

GTA AAG GCA GCT AAA CCG GAG GCC ATA ATT GTA GGA GAG GTT TGGV K A A K P e A I I v G e v w

CAT OAT GCT TCT CCT TGG CTT AGA GGG GAT CAG TTT GAC AGT GTAH D A S P w L R G D Q P D S v

ATG AAC TAT CCT TTT AGA AAT GCG GTT GTG GAC TTT TTT GCA AAA

M N Y P P R N A V v D F A K

AGG AAA ATA AGT GCT TCT C0C TTT AAT ACA ATG ATT ACA GAG CAAR K I S A S R N T M I T Q

CTT ATG AGG CAT ATG GAT AGT GTA AAT AGG GTT ATG TTT AAT TTA

L M R H M D S v N R v f P N L

4'5 ATA GCA AGT CAT GAT ACT GAG AGO TTT TTG ACT TTA GCT AAT GGCI G S H D T E R P L T L A N G

90 ATG GTT GCG AGA ATG AAG TTA GCA TTA GTG TTT CAA TTT ACC TTTM v A R M K L A L v F Q P T P

135 GTT GGA ATC CCA TAT ATT TAC TAT GGG CAC GAG GTA GGA ATG GTGv G I P Y I Y Y G D 8 v G M V

180 GGA GAT TAT GAC CCT GAT TOC AGA AGA TGC ATG ATA TGG GAG GAGG D Y D P D C R R C M I H e e

225 GAA AAG CAA AAT AAA AGT ATT TTT AAT TTC TAT AAA AAG TTG ATTe K Q N K S I P N F Y K K L I

270 TCT ATA AGG AGA GAA AAT GAG GAG CTC AAA TAC GGA ACT TTC TGTS I R R e N e e L K Y G S F c

315 ACT TTA TAT GCT ATA GGA AGA GTG TTT GCT TTT AAG AGG GAA TACT L Y A I 0 R V P A P K R e Y

360 AAA GGT AAA TCA ATA ATT GTT GTA CTA AAT AAC AGT AGC AAG CAG

K G K S I I v v L N N S S K Q

405 GAA GTG ATA TTT TTG AAT GAA GTA GAA GGA AAA GAA GAT ATT TTA

e v I P L N e v E G K e 0 I L

450 AAG ATC AAG OAA TTA AAA AGA AGT GGA AAT CTC CTT TAC TTG CAAK K K e L K R S G N L L Y L Q

495 CCT AAC TCT GCC TAT ATT TTA AAC TAA CCA ATC TGA GTT TAA CGCP N S A Y I L K * * *

540 AAT AAT ATG AAG ATA GAA AAA TAT AGO GGA ATT AAA ATT CCC CTA

58 5 TAT TTT TTA AAA AAA ACA GC GTG ATG GTA TGA AAG ATA CAT CCC

630 GCT ATG GCA GAG ATT ATC AAT ATT ATA AGG ATT ATG AGA GAT TAT

ATG GAA GTT ACG ACA TTA CAA AGC TTA TCA CAT TAA AAA AAT TAG675

TTG AAG CAA GTG AAT ATG AAG AGT GGA AAG ATT ACA AAG AGT TCA720

AGG AAT GOT GTA ATT CTG AAG AAA GAC ATA ATT GGA AAA ATT CAC765

CTA AAT ATA TTG AAT GGG AAA AG810

855

900

945

990

1035

1080

1125

1170

1215

1260

1305

1350

1395

1440

1485

1530

1575

1620

1665

FIG. 2. Nucleotide sequence of the CDase gene and deduced amino acid sequence. The putative -35 and -10 regions and the

Shine-Dalgarno (SD) sequence are underlined and double-underlined, respectively. The arrows indicate regions of dyad symmetry. The stop

codon of the CDase gene and the two closest stop codons in the same reading frame are designated with asterisks.

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1710

1755

1800

1845

1890

1935

1980

2025

2070

2115

2160

2205

2250

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2340

2385

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CDX C. th. 166 FFGGDLQGIIDKIDYLKDLGINAIYLTPIFLSHSTHKYDTTDY 208APL C. th. 416 FFGGDLKGIDDKLDYLKSLGISVIYLNPIFQSPSNHRYDTTDY 458NPL B. st. 170 FFGGDLQGIIDHLDYLVDLGITGIYLTPIFRSPSNHKYDTADY 212

1________ 2

CDX C. th. 238 DAVFNH.......... 320 DGWRLDVANEAPL C. th. 488 DGVFNH .......... 594 DGWRLDVANENPL B. st. 242 DAVFNH .......... 323 DGWRLDVANEAMY A. or. 117 DVVANH .......... 201 DGLRIDTVKHAMY B. am. 98 DVVLNH .......... 226 DGFRIDAAKHAMY B. li. 129 DVVINH.......... 255 DGFRLDAVKHAMY B. st. 101 DVVFDH .......... 230 DGFRLDAVKHIAM P. am. 291 DVVYNH .......... 369 DGFRFDLASVCGT B. sp. 135 DFAPNH .......... 224 DGIRVDAVKHPUL K. ae. 600 DVVYNH.......... 670 DGFRFDLMGY

3 4 **

CDX C. th. 354 EVWHDASPWLRGDQFDSVMNYPF .... 416 LIGSHDAPL C. th. 627 ENWNDASLDLLGDSFNSVMNYLF 699 LLGSHDNPL B. st. 357 EIWHDAMPWLRGDQFDAVMNYPF ....419 LLGSHDAMY A. or. 230 EVLD ........ ............... 292 FVENHDAMY B. am. 261 EYWQ....................... 323 FVENHDAMY B. li. 289 EYWQ....................... 352 FVDNHDAMY B. st. 264 EYWS ........ ............... 326 FVDNHDIAM P. am. 454 EWSV.......................502 FIDVHDCGT B. sp. 268 EYHQ ........ ............... 323 FIDNHDPUL K. ae. 704 EGWD ........ ............... 827 YVSKHD

FIG. 3. Similarities in the amino acid sequences of CDase andvarious amylolytic enzymes. Enzymes and organisms are abbreviatedas follows. CDX, CDase; APL, a-amylase-pullulanase; NPL, neo-pullulanase; AMY, a-amylase; LAM, isoamylase; CGT, CGTase;PUL, pullulanase; C. th., C. thernohydrosulfwricum; B. st., B.stearothennophilus; A. or.,A. oryzae; B. am., B. amylolquefaciens;B. Hi., B. lichenifonnis; P. am., P. amyloderamosa; B. sp., theBacillus sp.; K. ae., K aerogenes. See the text for the references inwhich the sequences can be found. Numbering starts from the firstamino acid of the mature proteins. Numbered bars above the se-quences denote highly conserved regions among a-amylases (28).Catalytic and substrate-binding residues proposed for CDase areindicated with asterisks and bullets, respectively.

a monomer with a molecular weight of 68,028. This value isclose to that reported for the B. coagulans enzyme (62,000)(16), while the enzymes from the Bacillus sp. and B. sphaen-cus consisting of two subunits have larger molecular weights(126,000 and 144,000, respectively) (30, 43), and these arethe only Mrs reported for bacterial CDases to date. Previ-ously, we have reported about the unique thermoactivity andthermostability of the CDase partially purified from C.thennohydrosulfuricum 39E (35). The recombinant enzymepurified to homogeneity in this study shows the same prop-erties.The substrate specificity of C. thernohydrosulfuricum

CDase is very similar to that of the Bacillus sp. enzyme (43).Both enzymes hydrolyze a-, 1-, and y-CDs as well as linearmaltodextrins, with the exception of maltose. The ring sizeof a CD has a significant effect on the activity of the enzyme:the bigger the substrate size, the less the activity. But CDasefrom the Bacillus sp. has the fastest hydrolysis rate formaltotriose and maltotetraose, while the best substrate forCDase from C. thermohydrosulfuicum is c-CD.The final products from CDs and maltooligosaccharides

hydrolyzed by the enzyme are maltose and a small amount ofglucose. This is similar to the action of some other CDases.Maltose is the main product of CDase from the Bacillus sp.when CDs and short linear dextrins are used as substrates(43). Pseudomonas sp. a-amylase, which has high activityon CDs, produces maltose and glucose as the final productsfrom different substrates (14).The CDase from C. therinohydrosulfuiicum is inhibited by

thiol reagents (p-chloromercuribenzoate, Hg2+, Ag+, andCu2") and by N-bromosuccinimide, an indole-oxidizing re-

agent. From the CDase amino acid sequence, the enzymehas 6 Cys and 14 Trp residues. It remains to be determinedwhich of these residues may participate in the formation ofthe active site. The possible existence of a sulfhydrylgroup(s) in the active site of the enzyme was suggested forCDase from the Bacillus sp. (43), while CDase from B.coagulans was not inhibited by thiol reagents (16).As this is the first report of a CDase gene sequence,

comparison with other CDase genes was not possible.CDase was compared with some amylolytic enzymes withknown amino acid sequences. Nakajima et al. (28) havedemonstrated four highly conserved regions in a-amylasesfrom different organisms. Sequences similar to the first,second, and fourth regions have been detected in CGTase,pullulanase, neopullulanase, a-amylase-pullulanase, isoam-ylase, and CDase (19; this study). This finding is intriguingbecause CDs, substrates preferred by CDase, generallycannot be cleaved by other amylolytic enzymes and mayeven be inhibitory (26, 32). Nevertheless, the results pre-sented in Fig. 3 suggest that all of these enzymes may use asimilar mechanism of catalysis. The differences in substratespecificity and final products may be explained by thevariations in subsites (13) and their influential contribution inthe structure of the active sites.

In order to determine the possible functions of the CDaseresidues within the conserved regions, we used the molecu-lar model of Taka-amylase A (24). X-ray crystallographicanalysis has been carried out only for this enzyme, acomplete structural model has been built, and catalytic andsubstrate-binding residues have been proposed (6, 24). Inaccordance with the homology between Taka-amylase A andCDase, the amino acid residues which play an important rolein the enzymatic reaction catalyzed by CDase have beenproposed (Fig. 3). There are at least two points to suggestthat such a comparison may be fruitful. First, on the basis ofthe amino acid sequence homology between Taka-amylase Aand CDase and the development of a structural model forTaka-amylase A, the residues corresponding to the putativeactive center of neopullulanase from B. stearothermophilus(20), CGTase from the Bacillus sp. (29), and amylopullula-nase from C thermohydrosulfuricum 39E (23) were identi-fied and altered by means of site-directed mutagenesis. As aresult, the enzymes either completely lost activity (when acatalytic residue was substituted) or changed product spec-ificity (as demonstrated for neopullulanase) when a sub-strate-binding residue was altered. It is noteworthy that theproposed Taka-amylase A catalytic residues are conservedboth in the enzymes mentioned above and in the C. thermo-hydrosulfuricum 39E CDase. Secondly, CDs can be de-graded by Taka-amylase A; although the rate of hydrolysis ismuch lower than that for amylose (36), it does suggest acommon catalytic mechanism between these enzymes.Moreover, by using structural modelling, 1-CD was shownto fit well in the model of Taka-amylase A at the bottom ofthe cleft in which residues involved in the active center arelocated (24).

ACKNOWLEDGMENTS

We owe special thanks to Saroj Mathupala for helping in theanalysis of DNA and protein sequences and to Susan Lowe for hervery helpful comments regarding this article. We are grateful toBadal Saha for valuable advice and discussions.

This research was supported by the Cooperative State ResearchService, U.S. Department of Agriculture, under agreement 90-34189-5014.

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CYCLODEXTRINASE GENE FROM C. THERMOHYDROSULFURICUM 5405

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