a-Amylase from the Hyperthermophilic Archaebacterium

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 32, Issue of November 15, pp. 24402-24407,1993 Printed in U.S.A.

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

a-Amylase from the Hyperthermophilic Archaebacterium Pyrococcus furiosus CLONING AND SEQUENCING OF THE GENE AND EXPRESSIONIN

ESCHERICHIA COLT*

(Received for publication, August 24, 1992, and in revised form, July 28, 1993)

Kenneth A. Ladermant, K. Asadat, T. Uemorit,H. Mukai5, Y. Taguchit, I. Kato§, and Christian B. AnfinsenSll From the $Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and STakara Shuzo Co., Seta 3-4-1, Otsu, Shiga 520-21,Japan

A gene encoding a highly thermostable a-amylase from the hyperthermophilic archaebacterium P y r o coccus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, whichcorresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine. The amylase expressed in E. coli exhibits temperature-dependent activation characteristic of of the original enzyme from P. furiosus, but has a higher apparent molecular weight which is attributed to the improper formation of the native quaternary structure. No homology was found with previously characterized promotor or termination sequences. The deduced amino acid sequence displayed strong homology to the a-amylase A of Dictyoglomus thermophihm, an obligately anaerobic, extremely thermophilic bacterium. Evolutionary implications of this homology are discussed.

Hyperthermophilic archaebacteria provide an extraordinary opportunity to study the factors influencing protein thermostability. Unfortunately,due to therecentness of their discovery, the extent of the research completed in this area remains limited. Pyrococcus furiosus is an anaerobic marine heterotroph with an optimal growth temperature of 100 “C, isolated by Fiala and Stetter (1986) from solfataric mud off the coast of Vulcano island, Italy. a-Amylase activity has been reported in the cell homogenate and growth medium of P. furiosus (Brown et al., 1990; Koch et al., 1990),and theenzyme has been purified to homogeneity (Laderman et al., 1993). The amylase is a homodimer with a molecular mass of 130 kDa which exhibits optimal activity at the optimal growth temperature of the organism. In an attempt tobetter understand the mechanisms of the enzyme’s inherent thermostability the gene coding for the a-amylase from P. furiosus was * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted to the GenBankTM/EMBL Data Bank withaccession numbeds) L22346. ll To whom correspondence should be addressed Dept. of Biology, The Johns Hopkins University, 34th and Charles Sts., Baltimore, MD 21218. Tel.: 410-516-8552;Fax: 410-516-5213.

cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. In addition the 3’- and 5”noncoding regions were analyzed in an attempt toidentify sequences involved in transcriptional regulation, and a search for homology between the deduced amino acid sequence and other a-amylases was completed. MATERIALS ANDMETHODS

Bacterial Strains-Cultures of P. furiosus (DSM 3638) were grown as described previously (Ladermanet al., 1993). For cloning and expression of the a-amylase gene, E. coli strain JM109 lrec Al, A(1acpro AB), end Al, gyr A 96, thi-1, hsd R 17, re1 Al, sup E 44,lF’tra D 36, pro AB’, lac IqZAM 151 was used. Plasmids, Enzymes, and Chemicals-The vector pUC18 was used for cloning and DNA sequencing. For expression the vector pTV118N from Takara Shuzo Co. (Kyoto, Japan) was used. Restriction endonucleases, alkalipe phosphatase, DNA Blunting Kit, Random Primer DNA Labeling Kit, DNA Ligation Kit, 7DEAZA Sequencing Kit, Mutan-K site-directed mutagenesis system, and SeaKem Ultrapure Agarose, 5-bromo-4-chloro-3-indolyl-~-~-galactopyranoside were obtained from Takara Shuzo Co. (Kyoto, Japan). Geneclean I1 Kit was obtained from Bio 101 (La Jolla, CA). PCR’ reagents and enzymes were from Perkin-Elmer Cetus. HybondN nylon hybridization filters and [Y-~’P]ATP were obtained from Amersham Corp. Ingredients for E. coli mediawerefromDifco. Isopropyl-8-D-thiogalactopyranoside (IPTG) andampicillin were obtained from Sigma. Ultrapure Urea was obtained from Bio-Rad. Assay of Amylase Activity and Native Polyacrylamide Gel Electrophoresis-The dextrinizing activity of a-amylase was determined at 92 “C using the 12/KI method as described previously (Laderman et al., 1993). One unit of the enzyme activity was defined as the amount which hydrolyzed 1 mg of starch/min. Native gel electrophoresis and subsequent staining techniques were performed as described elsewhere (Laderman et al., 1993). Preparation of Chromosomal DNAfrom P. furiosus-The cells were harvested and approximately 0.1 g of cells (wet weight) was suspended in 0.5 ml of 0.05 M Tris-HC1 (pH 8.0) containing 25% sucrose. To the suspension was added 0.1 ml of lysozyme (5 mg/ml). Incubation of the mixture for 1 h at 20 “C was followed by the addition 4 ml of SET solution (150 mM NaC1, 1mM EDTA, and 20 mM Tris-HC1 (pH 8.0)). 0.5 ml of 5% SDS and 100 pI of proteinase K (10 mg/ml) were added, and the mixture was incubated for 1 h at 37 “C. The solution was extracted with phenol-chloroform, and theDNA wasprecipitated with 2 volumes of ethanol. DNA was recoveredby winding and itwas rinsed in 80% ethanol. The yield of genomic DNA wasapproximately 1.3 mg from 0.1 g of cells. Preparation of a DNA Probe Using PCR-The NH2-terminal sequence of the intact a-amylase as well as a peptide fragment, determined previously (Laderman etal., 1993),were used for the construction of three degenerate oligonucleotide probes (Fig. 1). The probes were synthesized using an Applied Biosystems model380BDNA synthesizer. The PCR reactions were performed using 500 ng of P. furiosus ‘The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s). IPTG, isopropyl-P-D-thiogalactopyranoside;

24402

Cloning a-Amylase from of

P. furiosus

24403

JMlOS/pKENF-NH and deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, as ~ l y ~ s ~ ~ y ~ I l e A ~ n ~ h e I l ~ P h e G l y I l e H i 5 A s ~ i 5 G l n P r o L e uFERM G ~ Y A sBP-3782. n E. coli JM lOS/pKENF-NH cells were grown for 5 h at 37 "C in 5 5' ml of L broth containing 100 pg/ml ampicillin. The lac promotor was GATAAAATTAATTTTATTTT subsequently induced by the addition of 1 mM IPTG. The cells were C G C C C C allowed to grow for an additional 12 h, with vigorous shaking, then A A TGGTATTCATAATCATCAACC collected by centrifugation (7000 X g for 10 rnin), suspended in 200 C C C C C C G pl of 50 mM Tris-HC1, pH 7.0. The mixture was sonicated and A A centrifuged (30 min at 27,000 X g). The supernatant was incubated G 3' for 10 min a t 99 "C and centrifuged again. This supernatantwas used 1 as the crude cell extract. U e r 2 The a-amylase activity was measured using the standard activity assay described above. Computer Analysis-The search for existing sequences displaying homology to the P. furiosus a-amylase gene was completed through nus of a PeDt1.de F r a m . a € GenBank'" using FASTA searches. Predictions of secondary structure and physical characteristics based on deduced amino acid sequence were completed using PC/GENE (IntelliGenetics Inc., Mountain ThrLeuAsnAspMetArgGlnGluTyiryrPheLyS View, CA) or the Wisconsin Genetics Computer Group (WGCG) 3' sequence analysis software package Version 6.0 (Genetics Computer TTACTATACTCAGTTCTTATAATAAAATTTT Group, University of Wisconsin Biotechnology Center, Madison, WI). G G G G C C G G G C

m2zkmiw.s of

t h e I n r a m EnZvm

T

C 5'

RESULTS

Cloning and Sequencing of the a-Amylase Gene: Characteristics of Coding and Noncoding Regions-The preparation of a probe using nested PCR resulted in the amplification of a DNA fragment of approximately 1 kilobase. The amplified DNA fragment was blunted using the DNA Blunting Kit (Takara Shuzo, Kyoto) andsubcloned into the HincII siteof pUC18 (Sambrook et al., 1986).The cloned plasmid was sequenced by the dideoxy method. chromosomal DNA as the template with 100 pmol of primer 1 and When a Southern blot prepared with digestedgenomic DNA 100 pmol of primer 3 (see Fig. 1).The PCR profile was 94 "C for 0.5 P. furiosus was probed with the 32P-labeled PCR product, from min, 40 "C for 2 min, and 72 "C for 2 min. Amplification was continued for 35 cycles in a total volume of 100 pl. 1 p1 of the reaction a 5.3-kb PstI fragment, a 3.1-kb HindIII fragment, a 5.3-kb mixture was further reamplified with primer 2 (see Fig. 1) and primer XhoI fragment, and two EcoRI fragments of 0.7 and 2.4 kb 3. The PCR profile was same as above, but the number of cycles was were found tospecifically hybridize to theprobe. 30.Five microliters of this mixture were analyzed by agarose gel Three clones carrying an identical 5.3-kb PstI fragment electrophoresis. were identified by colony hybridization of an enriched gene Cloning and Sequencing of thea-Amylase Gene of P. furiosus-5pg portions of P. furiosw chromosomal DNA were digested with PstI, bank of P. furiosus genomic DNA using the PCR product HindIII, XhoI, or EcoRI. The resulting fragments were separated on known to contain the coding region for the protein's NH2a 1%agarose gel, transferred to a nylon membrane, and hybridized terminal sequence. One of these clones, shown to contain the to the random primer labeled PCR product (Sambrook et al., 1986). a-amylase coding region in the same orientation as the vectorHybridization was carried out for 2 h at 65 "C in 6 X SSC, 0.1% SDS, derived lac promotor, was digested with HindIII and allowed 5 X Denhardt's solution, 100 pg/ml calf thymus DNA, and 1 X lo7 to self-ligate removing a portion of the 3"noncoding region. cpm/ml 32P-labeledprobe. The membrane was washed for 40 min in a solution of 2 X SSC and 0.1% SDS at65 "C and then for 20 min in The nucleotide sequence of the resulting 3.1-kb insert was determined in bothorientations.Therestrictionmapand a solution of 0.5 X SSC and 0.1% SDS at 65 "C. Genomic DNA digested with PstI was size-fractionated on a 1% sequencing strategy are shown in Fig. 2. The complete nuagarose gel, and a size-fractionated library, with an insert size of cleotide sequence of the 3.1-kb insert is given in Fig. 3. approximately 5-5.5 kb, was constructed inthe PstI site of pTV118N The a-amylase gene encompasses 1950 nucleotides,with and used to transform E. coli JM109 cells. Recombinant plasmids containing the target sequence were screened by colony hybridization the initiationcodon GTG at position715 (Fig. 3). There is no (Sambrook et al., 1986) using the 32P-labeledPCR product produced strong homology, preceding the coding region, with known as described above. The screening yielded three positive clones, and archaebacterial, eukaryotic, or eubacterial concensus promoone of these, pKENF, was used for further characterization. tor sequences described previously. Immediately upstream of Double-stranded recombinant plasmids and single-stranded DNA the coding region is the sequence GGTGGA, similar to the were isolated following the protocol of Sambrook et al. (1986). Sequencing was completed using the dideoxy termination method of putative ribosome-binding site of the glyceraldehyde-3-phosSanger et al. (1977) with the 7-DEAZA sequencing kit. Overlapping phate dehydrogenase gene of Pyrococcus woesei (GAGGT) C content of the a-amylase (Zwickl et al., 1990). The G subfragments were generated using the method of Yanisch-Perron et al. (1985). gene is 41.6%, slightly higher than the value reported for the Expression of P. furiosus a-Amylose Gene in E. coli-To prepare a total genome of 38% (Fiala and Stetter, 1986). As has been construct which expressed the P. furiosus a-amylase in E. coli an NcoI site was created at the translation initiation codon of the a- seen in other sequenced genes from extreme thermophiles, A position of the amylase gene using the site-directed mutagenesis system Mutan-K and T are the preferred bases in the third (Takara Shuzo, Kyoto), converting the original initiation codon from codons (Zwickl et al., 1990). GTG to ATG. This plasmid (pKENF-2N) was digested with NcoI The five transcripts of the Sulfolobus virus-like particle and self-ligated to eliminate the 5"noncoding region and toposition SSV-1 (Reiter et al., 1988b) and the glyceraldehyde-3-phosthe gene at a suitable distance from the vector-derived lac promotor and ribosome binding site. The resulting plasmid (pKENF-N) was phate dehydrogenase gene of P. woesei (Zwickl et al., 1990) digested with HindIII and self-ligated to delete a 3"noncoding region; include the sequence TTTTTT in a pyrimidine-rich region directly downstream of the terminationcodon. A pyrimidinethe product was then designated pKENF-NH. E. coli JM 109 cells carrying this plasmid were designated E. coli rich region exists 34 bases 3' of the termination codon in the 3

FIG. 1. Degenerate oligonucleotide primers based on the NH2-terminalamino acid sequence. Probes were designed for use in the PCR amplification of a portion of the P. furiosus a-amylase gene. The oligonucleotides were prepared as shown, based on the NHz-terminal sequence of the purified enzyme and a peptide fragment.

+

Cloning of a-Amylase from P. furiosus

24404 Base pairs

1000

2000

3000

-718

I

I

I

-658

-620

-680

c TGC

AGG GGA

Grr

r G c AAA GCTAAr

AGA ACT AGC TCA AGG TAA

-598

P

wLr

AAC TTC AGT AGC r G G TGG AGG AGG CGG AGG AAG

AAG AGA c a T AGA AAA AGC AAA AGA GGC AAT AGA ARR

-568 TCT CCT rcT -500 rX: m ATC TTT ATP -448 ACT TCC G rG GGC AGr -388 w.r m GAA G m ccc -328 GGA AAT rcr ATT TCG -268 CCC CAA CCG TCT TTT -200 GAT TCT TTT rcA rm -148 AGG ACT ATA ARA ATT

9 AGT T M

GGG

crc

r c T A m GCT

-530

.ur GGG

AGC

r a T ATT TTC

wc

ACT

GAT ACC TTT

Grr rcA

m GTG

ccc r c T

TCT GAT CTC CAG

S

S

ORF c-

- -- ""4-

"

c-

" C

" " "

"

"

4

-478 TCT CTG -418 TCC r c T -350 ArG AGA -298 TCCACT -238 TCC TTA -178 Gl'T CTC -110

E

Pst I EcoRl Hind 153 111

H. Hc. Hinc II S. Sac I

FIG. 2. Restriction map and sequencing strategy of the cloned PstI-Hind111 insert carrying the a-amylase geneof P . furiosus. Arrows indicate the individual sequence runs. ORF, open 333 P , PstI; E, EcoRI; H , HindIII; Hc, HincII; S, SacI. reading frame.

a-amylase gene, but unlike other archaebacterial sequences examined,there is nopyrimidine-rich region immediately downstream from the TAG stopcodon. Expression of P. furiosus a-Amylase Gene in E. coli and Comparison with the Enzyme Purified from P. furiosus-For expression of the P. furiosus a-amylase in E. coli an insert containing the gene flanked by archaebacterial noncoding regions was inserted in the expressionvector pTV118N. This construct,693 with the P. furiosus 5' sequence intact, was found to not express any thermophilic a-amylase activity. To prepare a more stream-lined construct for expression in E. coli, a unique NcoI restriction site was created at the initiation codon, converting the initiation codon from GTG to ATG, and the P. furiosus noncoding region was removed. The resultant expression plasmid, denoted pKENF-NH, placed the gene in the correct reading frameat an appropriate distance downstream of the vector promotor(Fig. 4). IPTG-induced E. coli JM109 cells transformed with the plasmid pKENF-NH were found to produce 0.2 unit/ml of thermophilic1053 amylase activity in the crude extract. The heterologously expressed amylase was compared with the enzyme purifiedfrom 1113P. furiosus regardingmolecularweight and temperature 1173 dependence of activity. When the apparentmolecular weights of the recombinant protein and the isolated enzyme were1233 compared on an activity stained native gel the protein produced in E. coli displayed a higher apparent molecular weight. The comparison of the temperature dependence of the a-amylase activity between the purified and re1353 combinant proteins displayed virtually identical relationships of relative activity asa function of temperature (Fig. 5). When the 1413 complete amylasegene was analyzed, no protein or nucleotide sequence was found which displayed complete homology with the deduced NH,-terminal sequence of the peptide fragment. Thissuggests that the peptidesequence, on 1533 which thesynthesis of primer 3 wasbased, represents a contaminant and not a portion of the P. furiosus a-amylase. It was therefore fortuitous that the degenerate primer preas a random primerfor the PCR reaction. pared was able to act To confirm that the a-amylaseexpressed in E. coli was the enzyme purified from the bacterium, not a unique enzyme with a similar activity profile and a shared amino terminal

TTT AGC GTAAAT

AAC

TAG M T

CTT

ArcrcA

ATG GCA AGC ACT AGA

TTT GGA A r c AGA AAT ATT TCA TAT

m

A m

TGG M C

TTG

rM

AAG TTG

rM mc rcG

GTG

cTr

m ACT

mr

r c A GCA

A m

Trr

TTC

CCA TTT ACC

TTT

AAA TCT GAG ACA A r T

Trr

TCC

TCA ACT RTT GTA ACTGTCCTG

TTT

ATG GTT GCA GGA GGC A r T TTT GCC ATTGCT

ACA GCT GTT ATT TTCTTG

-88

TCC ATG CTA

ACA CCC TGTAAT

GAG ATT TGG ATT TTC CTA TAT AWL AAG CCT TAG T T A

-58

-28

TTG AGC CAT TAA ATA TAT AAG GAA G r A TCA CTC

rrh

33

VA ATP. rm A GGA ATT CAC ~ MC CAT

glyasplys 63

TTT

-1 1 GTG ATT MT GGG r G G ACG GAA GTG

met

-

3 GGA

P.

TAA

m rAc

ile a m pheilephegly

CAG

&c c x

GGC AAC TTT GGA

i l e h l s asn h i s g i n p r o l e u q l y 93

a m p h eq l y

' K G GTG TTT GAG GAG GCTTAT GAA AAG TGT TAC TGG CCG TTT CTG GAG ACT CTG GAG GAA t r p v a l phe glu q l u a l a t y r glu l y s c y s t y r trp p r o p h e l e u glu t h r leu q l u g l u

123 TAT CCA AAC ATG AAG GTT GCC ATT CAT ACA AGT GGC CCC CTC ATT GAG TGG CTC CAA GAT t y r p r o asn met l y s v a l a l a i l e h i s t h r ser g l y p r o leu i l e glu trp l e u g l n a s p

183

213

AAT AGA

ccc

GAA TAC ATA GAC TTG CTT

asn arg p r o g l u t y r i l e a s p 243

am

AGT CTA GTG AAA AGA GGA CAG GTG GAG ATA

l e u l e u arq ser l e u val l y s arg g l y g l n v a l g l u i l e 273

GTC GTT GCT GGG TTC TAC GAG CCT GTG CTA GCA TCAATC

Val Val a l a g l y p h e t y r g l u p r o v a l l e u 303

CCA AAG GAA GAT AGA ATA GAG

a l a ser i l e p r o l y s g l u a s p

arg i l e g l u

CAG ATA AGG T T A ATG AAA GAG TU: GCT M G AGT ATT GGA TTT GAT GCT AGG GGA GTT TGG

l e u met l y s g1u t r p a l a l y s ser i l e g l y p h e a s p a l a arq g l yv a 1 trp 393 GAG CTC GTA M G ACC CTT AAG GAG AGC GGA ATA GAT arq v a l trp g l n p r o glu l e u Val l y s t h r leu l y s g l u ser g l y i l e a s p 453 GTT GAC GAT me CAC r T c ATG & G r GCG GGA TTA AGT AAA GAG GAG crG TAC val a s p a s p t y r h i s p h e met ser a l a g l y leu ser l y s g:u q l u l e u t y r 513 TGG CCA TAT TAT ACG GAA GAT GGT GGG GAA GTT ATA GCT GTT TTC CCGATA GAT GAG AAG trp p r o t y r t y r t h r q l u a s p g l y g l y g l u v a l i l e ala val pheproileaspqlulys

g l n i l e arg 363 ACT GAA leu t h r g l u 423 TAT GrA a m t y rv a l i l e 483 CTA

AGA GTA TGG CAA CCA

543

573 AGA TAT r T G ATT ccc rTr AGA ccc G r T GAT AAG GTC TrA GAA r A c c r G CAT TCT crc l e u arg t y r l e u i l e p r op h e arq p r o v a l a s p l y s val leu glu t y r l e u h i s S e r l e u 603 633 AT?. wn GGT GAT GAG AGC AAA G n GCA GTP. T r T CAT GAC GAT mr GAG AAG m GGA ATC ile aspglyasp glu ser l y s val a l a v a l p h e h i s a s p a s p g l y g l u l y sp h eg l y ile 663 TGG CCT GGA ACT TAT GAG TGG GTG TAT GAA AAG GGA TGG T T A AGA GAA TTC TTT GAT AGA trp p r o g l y t h r t y r g l u trp v a 1 t y r g l u l y s g l y trp l e u arg g l u p h e p h e a s p arg 723 153 ATT TCA AGT GAT GAA AAG ATA AAC TTA ATG CTT TAC ACT GAR me T r A GAA AAA rm AAG i l e ser ser a s p g l u l y s i l e a m leu m e t l e u t y r t h r g l u t y r l e u glu l y s t y r l y s 703 813 CCT AGA GGT CTT GTT TAT CTT CCA ATA GCT TCA TAT "'7 GAG ATG AGC GAA TGG TCA TTG p r o arg q l y leu val t y r l e u p r o i l e a l a 5er t y r p h e q l u met ser glu L r p Ser l e u 043 073 CCA GCA AAG CAG GCA AGG CTC TTT GTG GAG TTCGTCRAT GAG CTT AAA GTT AAA GGT ATR p r o ala l y s g l n a l a arg leu phevalgluphe val asn glu l e u l y s Val ~ Y SqlY ile 903 933

TTG

mr

GAA AAG TAC AGG GTA TTT GTT AGG GGA GGA A T rr G G

phe q l u l y s t y r 963

AAG

a r

TTC TTC r m

AM

mc

arq v a lp h ev a l

arq q l y qly i l e t r p l y s a m p h ep h et y r 1YS LYr 993 CCA GAG AGC AAC TAC ATG CAC AAG AGA ATG CTA ATG GTA AGT M G TTA GTG AGA AAC AAT p r o g l u ser asn t y r met h i s l y s arg met l e u met v a l ser l y s leu v a l arg asn a m 1023 CCT GAG GCC AGG AAG TAT CTGCTG AGA GCA CAR TGT AAC GAT GCT TAT TGG CAC GGC CTC p r o q l u a l a arq l y s t y r l e u l e u arq a l a g l n c y s asn a s p a l a t y r t r p h i s glY l e u 1083 TTC GGT GGA GTA TAT TTA ccc CAT CTT AGG AGG GCC ATC TGG AAC AAT TTA A r c M G GCC t y r leu p r oh i sl e u arg arq a l a ile t r p asn asn l e u i l e I Y Sa l a phe g l y q i y 1143 MC AGC TAT GTA AGC CTT GGA AAGGTC ATA AGG GAT ATC GAC TAC GAT GGC T T T GAG GAA a m ser t y r v a l ser l e u q l y l y s val i l e arg a s p i l e a s p t y r a s p g l y p h e q l u qlu 1203 GTT CTC ITA GAG AAT GAC AAC TTT TAT GCA GTG m AAA CCC TCT TAC GGT GGT TCC TTG v a l leu i l e glu a s n a s p a m p h e t y r a l a Val p h e l y s p r o 5eI t y r 9lY9lY Ser l e u 1293 1263

GTG GAG TTT TCA TCA AAG AAT AGA CTC GTG ARTTAT GTA GATGTTCTG GCA AGA AGG TGG v a l glu phe S ~ ser T l y s asn arq l e uu a l asn t y r v a l a s p Val l e u a l a ar9 arg t r p 1323 GAA CAC TAT CAT GGC TAT GTG GRR AGT CAA m GAT GGA g l u h i s tYTh i s g l y t y r v a l glu ser gln p h e a s p g l y 1383 GAG AAA AAG ATA CCA W T GAA RTA AGA AAA GRR GTT GCT q l u l y s lYs iie pro a s p g l u i l e arq l y s g l u val a l a 1473 1443 ATG CTT CAA GAT CACGTAGTCCCCCTG GGA ACA ACT CTG met l e u asp h ivsa i l e u g ltyht rh r leu 1503

GTA GCC AGC ATT CAT GAG CTC

val a l a ser i l e h i s g1u leu

TIC GAC AAG TAC AGAAGG TTC tyrasplystyr a w a w phe TTC A T G TTCTCA AGA glu a spph e met phe ser arq GAR GAC

FIG. 3. Nucleotide and deduced amino acid sequence of the a-amylase gene. The nucleotide sequence from the PstI site, 717bp 5' of the initiation codon, to the HindIII site atposition 2423 is presented. The underlined portion represents thenucleotide sequence coding for the NHp-terminal amino acids upon which the oligonucleotide primerswere based. The single underlined region represents the sequence homologous toprimer 1. The double underlined region represents the sequencehomologous to primer 2.


24405

Arc GGA GAG m ccr AGG GTT CCA TAC rcA r m CRA CTA CTA GAT GGU GGA gln gin glu lie giy glu phe pro arg vai pro tyr ser tyr g l u leu leu asp gly gly 1563 1593 ATA AGG CTG AAG AGG GAA CAC T T G GCA UTA GA4 G T T GAR ?+E3 ACA GTG ARG TTA GTG ART CRA CAG GAG

ile ar9 leu lys 1623 GAT GGA rrT GAG q l y phe glu 1683 FM; wv\ CTT ARC p l u leu asn 1143 GAA ATT GTC GTT glu i l e v a l v a l 1803 ArG G A GTC ~ TGG met q l u v a l trp 1863 ATC CAG CAG GGT ile gln gln gly 1923 -A

AAR

m

leu lys phe 1983 CTC CCT CAT 2043 TTA CAA RAT

2103

KT TTC ccc

qlu his leu qly ile glu val glu lys thr v a l lys l e u Val asn 1653 GTG GAG TAT ATA GTG AAC ARC ARG ACA GGA ART CCT GTP. TTG TTC GCA val glu tyr ile val asn asn lys thr gly asn pro Val leu phe a l a 1113

GTT

GCA GTT CAG AGC ATA ATG GAG AGC CCA GGA GTT CTA A m GGG v a l ala v a l qln ner ile met slu ser pro caly val l e u a w gly ~ Y S

1173

GAT GAC AAG TAT Gca G11 GGG

AAG

TTT

asp asp lp. tyr ala val gly lys phe 1833 ARG TAT CCA GTA AAG ACT CTC AGT CAA lys tyr pro v a l lys thr leu ser gln 1893 GTC AGC TAC ATA GTT CCA ATA A M ; "G v a l ser tyr ile val pro ile arg leu 1953 GAG GAR GCC rcG GGA TAG GGA GGC CCT CAT glu glu a l a Ser gly AHB 2013 CGG ccc TTC TAT TTT ATT T T R AAC GTC RAT 2073 GAA CAA Arc TCT CCA CTT GCG GGC ATT CCU 2133 TTC ACT TCT GGC TCG ARR UGT m TTC TTT 2193 GTT CCA GGC CTT TTT TCC TCC AAT TCA T T G 2253 CCT CTT GCu TAA GGA CAC TCC TCT ACT RTG 2313 UCA ACT TCC CTC TCU GTT AAT TCG TAGU G R 2373 CCT GGG AGC UGA GGA CCT CCC T T A GCC AGG

2163 TTG AAC TTT 2223 GTT GTC GCA 2283 GCA TUG GCU 2343 TTT CCT TCC 2403 AAG TTG Trc A x AGA AAG

err

FIG.3"Continued

C X AAG W T GAR GAC GAR ala leu lys phe glu asp glu GCA

AGT GA4 AGT GGC TGG GAT CTA ser g l u se= g l y trp asp l e u

GAT I\AA ATU AGG TTT glu asp lys ile a w phe ~ Y S

GAG

CAC CAR T C A GGG

I

+ Ncol site(Mutan K. Takara)

CCC GAA AGA

GGT TT.4 CCA AGT Tn' CAR RRC CAT ATC

TTG CAC TCT TTG AGG

CTT

AGG AAT

AGU

ACT TCC TTC ATG TCA AGU

Ncol digestion (dilution )

.Hlndlll pKENF4

CCT CTC ACG A A G

TAC TCC ART CCA ACG GCA ATG

GGT TTG ATC TTC TTT ACG RRC TAC TCT GTR TTC CAG TGG AGT

1

Hind 111 digestion (dilution ) Self ligation

-

n

sequence, thephysicalcharacteristics of the protein were I""" examined. The gene product and the P. furiosus a-amylase were foundto have comparable isoelectric points, specific (Circular maps are n o t drawn in scale.) activities, and pHof optimal activity (data not shown). Primary Structure of P. furiosus a-Amylase and Computer Aided Comparison with Enzyme Homologs f r o m Mesophilic and ThermophilicSources-The deduced amino acid sequence of the P. furiosus a-amylase comprises649 amino acids, with a calculated molecular mass of76.3 kDa. This agrees well mu with the apparentmolecular mass of the protein, determined FIG.4. The construction scheme for the recombinant plasby gel electrophoresis under denaturing conditions, of 66 kDa mid pKENF-NH, used for theexpression of the P. furiosus a(Laderman et al., 1993). amylase gene in E. coli. The expression plasmid was constructed It is known that the amylaseof P. furiosus is secreted into by the introduction of a NcoI site at the initiation codon of the et al., amylase gene, followed by restriction digestion with this enzyme and thegrowth medium undernativeconditions(Koch subsequent self ligation. The resulting construct positions the initia1990). When the primary sequence of the proteinwas analyzed tion codon adjacent to the Shine-Dalgarno sequence of the plasmid using the PCIGENE PSIGNAL program, no typical eubac- lac promotor. The constructwas completed by digestion with Hind111 terialoreukaryoticNHz-terminalsignalsequences were followed by self-ligation, to removea portion of the 3"noncoding found. Using the standard activity assay, it was not possible region. to detect any thermophilic amylase activity in the extracellular media of JMlOS/pKENF-NH cell, confirming the ab- known t o be located in the active center and participate in sence of a signal sequencewhich would make the protein substrate binding in a number of amylases from a variety of competent for exportin E. coli. WhentheNH,-terminal sources (Bahl et al. 1991; Tsukagoshi et al., 1985), was persequence of the native enzyme purified from P. furiosus was formed no significant homology was found. comparedwiththepredictedNH2-terminal sequence they The codon usage of a-amylases from three thermophilic were found to be identical, suggesting there is no NH2-ter- sources, P. furiosus, D. thermophilum(Fukusumi et al., 1988), minal processing. and Bacillus stearothermophilus (Tsukagoshi et al., 1985), The GenBank'" FASTA searches resulted in the acquisitionwere compared. As noted previously, the higher the optimal of only a single strongly homologous protein sequence. The temperature of activity the more extensive the bias against query with the protein sequence of the P. furiosus a-amylase, the usage of the dinucleotide CG (Zwickl et al., 1990). This using the methodof Pearson and Lipman (1988), identified a trend is apparent notonly for the argininecodons, but for the 41.9% identity ina 537-amino acid overlap in thesequence of serine,proline,threonine,andalanine codons as well. No one of the a-amylases from the extremely thermophilic bac- other anomalies in codon usage attributable to thermostability terium Dictyoglomus thermophilum designated a-amylase A are apparent other than shifts inherent to the changes in (Fukusumi et al., 1988). The complete sequence of the D. amino acid composition. thermophilum a-amylase and a number of additional a-amyThe D.thermophilum a-amylase A displays physical charlases from various sourceswere aligned using the PC/GENE acteristics similar to those observed with the P. furiosus a CLUSTAL multiple sequence alignment program, and none amylase. The D.thermophilum enzyme exhibits optimal acdisplayed the high homology noted above. When a search for tivity at 90 "C with approximately 70% residual activity folhomology with previouslyidentified consensus sequences, lowing incubation for 1 h at this temperature (Fukusumi et

24406


FIG. 5. Comparison of the temperature-dependent amylase activity of the P. furiosus a-amylase and the amylase activity of the heattreated crude extract from pKENFNH-transformed JM109 E. coli cells. Amylase activity was determined

5* J

2

P. turiosw JMlO%KENF-NH

E

at a variety of temperatures, using the standard technique.

20

4800

100

60

120

140

Temp C

al., 1988). In contrast, the P. furiosus amylase is optimally active at 100 "C and exhibits a substantially higher thermostability: 85% after 3 hat 100 "C (Laderman et al., 1992). The variance in temperature-dependent activity and thermostability between two proteins displaying a high level of homology provides a unique opportunity to investigate the aspects of the primary sequence which confer enzyme thermostability. Computer analysis of the primary structures and the predicted secondary structures were prepared for the a-amylases from P. furiosus and D. thermophilum in an attemptto identify possible factors effecting thermostability. When hydropathy of the two sequences was plotted, using the PC/ GENE SOAP program, no apparent increase was found in the overall hydrophobicity associated with the increase in the thermostability of the P. furiosus amylase. When regions of high primary sequence homology were compared, no specific trend in hydropathy was noted. When the predicted secondary structures of the two proteins (obtained using the PC/GENE GARNIER program) were compared, little similarity in the proposed structures was noted. This dissimilarity was seen both overall and in areas of high primary structure homology. It is not possible to deduce, with confidence, the protein's secondary structure based exclusively oncomputer analysis. These resultsindicate that the primary sequence motifs upon which the computer secondary structure predictions are based differ sufficiently between the two proteins. DISCUSSION

A number of unusual characteristics of the P. furiosus aamylase gene set it apart from a majority of the genes characterized to date. The gene utilizes the relatively rare initiation codon GTG, as does the glyceraldehyde-3-phosphate dehydrogenase gene from P. woesei (Zwickl et al., 1990). It is possible that this represents a tendency in the usage of this initiation codon in hyperthermophilic archaebacteria. Unfortunately the number of structural genes isolated from these sources are too limited to allow an accurate assessment of whether the initiation codon GTG is in facta preferred initiation codon in these organisms, although this may be an intriguing possibility. It isknown that the production of a-amylase from P. furiosus is increased by the presence of starch(datanot

shown) indicating that the gene possesses an inducible promotor, a feature previously uncharacterized in hyperthermophilic archaebacteria. When the 5"noncoding region of the amylase gene was compared with the promotor sequences of other archaebacteria, no homology with the consensus sequences previously identified in Sulfolobus and Methanococcus (Reiter et al., 1988a) was found. The lack of homology with the putative ribosome-binding site of the P. woesei glyceraldehyde-3-phosphate dehydrogenase gene suggests eithera difference in the 3' terminus of the 16 S rRNA between the two closely related species, or adifferent promotor mechanism or both. In addition the P. furiosus amylase gene lacks the pyrimidine-rich region found immediatly downstream of the proteins3'termini in the forementioned archaebacterial genes. This lack of homology with previously investigated archaebacterisl promoters and terminationsequences may be a result of the local environment of the gene. The P. woesei glyceraldehyde-3-phosphate dehydrogenase gene is flanked closely by a number of open reading frames, whichwould benefit from atranslational coupling mechanism. In two instances with the SSVl genes in Sulfolobus, shown to share similar termination sequence characteristics with the P. woesei gene, this linkage between termination and re-initiation was observed. The P. furiosus gene exhibits no flanking open reading frames within the insert which was sequences, therefore precluding the necessity for translational coupling. This characteristic aswell as theinducibility of the gene are unique among the archaebacterial genes investigated thus far. The a-amylase from P. furiosus is one of a number of thermophilic proteins which have been expressed, in mesophilic hosts, in an active form. The three forms of a-amylase from D. thermophilum (Fukusumi et al., 1988), xylan-degrading enzymes from Caldocellum saccharolyticum (Luthi et al., 1990), andthe glyceraldehyde-3-phosphate dehydrogenase from P. woesei (Zwickl et al., 1990), produced under in vivo conditions at 73, 70, and 100 "C,respectively, have all been successfully expressed in E. coli. In these instances, the proteins produced by the transformed bacteriaremained inactive until they were heated to thetemperature appropriate to the enzyme's native conditions. This suggests that the proper folding of the protein into a structurewhich can be temperature-activated is possible at temperatures other than those at which the protein is produced under native growth conditions.


24407

evidence of their relationship. The P. furiosus a-amylase which is a dimer differs from the save enzyme activity remains as aforementioned examples in that it additionally requires the It is possible, since archaebacteria areconsidered to represent formation of an appropriate quaternary structure. Although a primitive kingdom, that the P. furiosusa-amylase,and temperature-dependent amylase activity was observed in E. therefore theD. thermophilum amylaseA, may be anexample coli, the apparent nativemolecular weight of the enzyme was of an archaic form of the enzymewhich is well suited to D. thermophilum amyhigher than the form purified from P. furiosus, suggesting extreme temperatures. In contrast, the lases B and C contain regions known to be well conserved in improper subunit assembly. It is not possible,however, t o determine whether this improper assembly is due to transla- several Bacillus species, hog, mouse, andhumanamylases form tion at lower temperature or to unidentified aspects of pro- (Fukusumi etal., 1988), and they represent the common of the enzyme, various examples of which are active over a duction in E. coli. Perhaps the most interesting facet of this research are the wide range of temperatures. REFERENCES evolutionary implications of the sequencehomology between the a-amylasesof P. furiosus andD. thermophilum. Based on Bahl, H., Burchhardt, G., Spreinat, A., Haeckel, K., Weinecke, A,, Schmidt, B., and Antranikian, G. (1991) Appl. Enuiron. Microbiol. 5 7 , 1554-1559 molecular phylogeny using rRNA sequences, existing orgaBrown, S. H., Constantino, H. R., and Kelley, R.M. (1990) Appl.Enuiron. nisms are seen to fall into three coherent groups eukaryotes, Microbiol. 5 6 , 1985-1991 Fiala, G., and Stetter, K. 0.(1986) Arch. Microbiol. 145,56-61 eubacteria, and archaebacteria (Fox etal., 1980). Substancial Fox, G. E., Staekebrandt, E., Hespell, R. B., Gibson, J., Maniloff, J., Dyer, T. A., Wolfe, R. S., Balch, W. E., Tanner, R. S., Magnum, L. J., Zableno, L. B., physiological and structuraldifferences exist between archaeBlakemore, R., Gupta, R., Bonen, L., Lewis, B. J., Stahl, D. A., Laehrsen, K. bacteria and eubacteria, which is evidence of their deep evoR., Chen, K. N., and Woese, C. R. (1980) Science 209,457-463 lutionaryseparation (Woese,1985). The phylogenetic tree Fukusumi, S., Kamizono, A., Horinouchi, S., and Beppu, T. (1988) Eur. J. Eiochem. 174,15-21 prepared by Pace et al. (1986) places archaebacteria closer to Koch, R., Zablowski, P., Spreinat, A., andAntranikian, G. (1990) FEMS Microbiol. 7 1 , 21-26 the common ancestorof all thekingdoms than one or bothof Laderman, K.Lett. A., Davis, B. R., Krutzsch H. C., Lewis, M. C., and Anfinsen, C. the other primary kingdoms, suggesting that archaebacteria B. (1993) J. Biol. Chem. 2 6 8 , 24394-i4401 E., Love, D. R., McAnulty, J., Wallace, C., Caughey, P. A., Saul, D., and are more primitive than one or both of the other lines. D. Luthi, Ber quist, P. L. (1990) A p l Enuiron. Microbiol. 5 6 , 1017-1024 thermophilum is a Gram-negative, obligately anaerobic, ex- Pace, R., Olsen, G. J., angwoese, C. R. (1986) Cell 45,325-326 W. R., and Liprnan, D. J. (1988) Proc. Natl. Acad. Sei. U. S. A . 8 5 , tremely thermophilic bacterium (Saiki et al., 1985). It shares Pearson, 2444-2448 with P. furiosus a low G + C content and a tolerance for Reiter, W., Palm, P., and Zillig, W. (1988a) Nucleic Acids Res 16, 1-19 Reiter, W., Palm, P., and Zillig, W. (1988b) Nucleic Acids Res. 1 6 , 2445-2459 extreme thermal conditions, but is a member of a different Saiki, T., Kobayashi, Y., Kawagoe, K., and Beppu, T. (1985) Int. J. Syst. Eacteriol. 3 5 , 253-259 phylogenetic kingdom. D. thermophilum has been shown to Sambrook, J., Fritsch, E. F., andManiatis, T. (1986) Molecular CloninA produce three different species of a-amylase, which can be Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Hagor, NY classified into two separate classes. First, amylase A, which Sanger, F.,Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci.U. S. A. displays a high degree of homology with the P. furiosus a7 4 , 5463-5467 H., Kondo, K., and Narita, K. (1982) Proc. Jpn. Acad. 58,208-212 amylase, andsecond, amylase B and amylaseC , which display Toda, Tsukagoshi, N., Iritani, S., Sasaki, T., Takernura, T., Ihara, H., Idota, Y., Yarna ata, H., and Udaka, S. 11985) J. Bacteriol. 1 6 4 , 1182-1187 homology with Taka-amylase A (Toda et al., 1982). No sigWoese, 8. R. (1985) The Bacteria, Academlc Press, New York nificant homology exists between these two classes, suggesting Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33, 103119 that they representtwo independent gene families or a single Z&kl, P., Fabry, S., Bogedain, C., Haas, A., and Hensel, R. (1990) J. Eacteriol. family which diverged at a point so distant that no feature 172,4329-4338

k.