THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Swiety for Biwhemistry and Molecular Biology, Inc. Vol. 268, No. 22, Issue of August 5, pp. 16332-16344,1993

Printed in U. S. A.

Sequencing of the Amylopullulanase (apu) Gene of Therrnoanaerobacter ethanolicus 39E, and Identification of the Active Site by Site-directed Mutagenesis"

(Received for publication, September 9, 1992)

Saroj P. MathupalaSEi, Susan E. LoweSq, Sergey M. PodkovyrovS, and J. Gregory Zeikus$jj** From the $Department of Biochemistry and the IlDepartment of M ~ r o b i o ~ # and Pubblic Health, M ~ h i g a n State ~ n i v e r s i ~ , East Lansing, Michigan 48824

The complete nucleotide sequence of the gene encod- ing the dual active amylopullulanase of 17rermoana- erobucter ethanolicus 39E (formerly Clostridium ther- m o h y d r o s ~ ~ r i c u m ~ was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of a-amylases and a-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M, 100,000 pro- tein that maintained the dual activities and thermosta- bility of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylol- ytic enzymes. Asp'", Glua2*, and Asp7- were individ- ually modified to their respective amide form, or the alternate acid form, and in all cases both a-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amy- lopullulanase as a new type of amylosaccharidase.

Enzymes harboring pullulan~e and a-amylase activity have recently been reported from various bacteria (Sakano et al., 1982; Coleman et al., 1987; Plant et al., 1987; Takasaki, 1987; Melasniemi, 1988; Saha et al., 1988, Sata et al., 1989; Spreinat and Antranikian, 1990). The dual activity of an amylase from Bacillus subtilis is reported to be due to two individual enzymes forming a complex dimer of 450,000 (Tak- asaki, 1987). An amylase-pullulanase enzyme of M, 220,000

* This work was supported by the Cooperative State Research Service, United States Department of Agriculture, under Agreement No. 90-34189-5014. 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 h a been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M97665.

tj Present address: Dept. of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205.

ll Present address: Bristol-Myers Squibb, Pharmaceutical Research Institute, P. 0. Box 5100, Wallingford CT 06492-7660.

** To whom correspondence should be addressed: Michigan Bio- technology Institute, Lansing, MI 48910. Tel.: 517-353-4674; Fax: 517-353-9334.

produced by Bacillus circulans F-2, which has a-amylase and pullulanase activities at equivalent rates, has been shown to contain two active sites for the individual activities (Sata et ai., 1989). a-Amylase-pullulanase from Clostridium thermo- hydrosulfuricum El01 has been reported to be structured similar to a cassette model, where half of the enzyme codes for a-amylase activity while the other half encodes pullulanase activity, based on sequence analysis studies (Melasniemi et al., 1990).

The amylopullulanase of Thermoan~robacter e t ~ n o l i c ~ 39E (formerly Clostridium thermohydrosulfuricum 39E (Lee et al., 1993) has been purified to homogeneity, and is a monomer ( M , 140,000), with a catalytic optimum of 90 "C @aha et al., 1988). Ca" is required for the stability of the enzyme, which has a half-life of 40 min at 90 "C (pH 6.0). Amylopullulanase is an enzyme exhibiting both a-amylase and pullulanase activities, giving rise to a-1,4 and a-1,6 glu- cosidic bond cleavage in starch and related polysaccharides (Mathupa~a et al., 1990). The gene encoding amylopuliulan- ase, designated apu, has been cloned and expressed in Esch- erichia coli and Bacillus subtilis.' In E. coli, the enzyme was located in the intracellular and periplasmic spaces, while in B. subtilis the recombinant enzyme was secreted to the culture supernatant. The expressed recombinant protein in E. coli had a M , of 160,000. The identity of the recombinant protein was verified by NH2-terminal amino acid sequencing, which was identical with the native enzyme. Nested deletion mu- tants constructed from the chromosomal DNA insert contain- ing the apu gene enabled restriction of the putative coding region to a size of 2.9 kbp: with concomitant decrease of the molecular weight of the expressed gene product from 160,000 to 100,000, without loss of dual activities or thermostability.

The purpose of this study was to sequence the apu gene and compare the deduced amino acid sequence with known amino acid sequences of a-amylases and debranching en- zymes, in order t o identify the conserved regions and the catalytic residues. Hydrophobic cluster analysis, a recently developed method based on two-dimensional representation of protein sequences, was used a means of comparing these enzymes. This is a more sensitive and effective method than linear alignment in that information is provided about the similarity of the secondary structures in proteins (Lemesle- Varlot et al., 1990). From these findings three amino acids were putatively identified as being involved in catalysis, and in order to test the hypothesis that a single active site imparts dual activity, site-directed mutagenesis of these amino acids -.

S. P. Mathupala and G. Zeikus, manuscript submitted The abbreviations used are: kbp, kilobase pairtsf; PAGE, poly-

acrylamide gel electrophoresis; rpm, revolutionslminute; kb, kilo- base(s); HCA, hydrophobic cluster analysis.

16332

Sequencing and Site-directed ~ ~ t u g ~ ~ e ~ ~ of apu Gene 16333

was performed and the effect on amylopullulanase activity determined.

MATERIALS AND METHODS

Reagents-All chemicals were of molecular biology or analytical grade and were obtained from Aldrich Chemical Co., or Sigma.

Bacterial Strains and Plasmids-E. coli strain DH5aF' (F- cg80dlocZAMl5 A(lacZYA-argF)U169 deoR recAl endAl hsdRl7(rK- mK') supE44 1- thi-1 gyrA96 relAl) was obtained from Bethesda Research Laboratories ( ~ ~ t h e r s b u r g , MD). E. coli TG-1 (supE hsdD5 thi A(lac-proAB) F' (traA36 proAB+ lacP lacZAM15)) was obtained from Amersham Corp. Phagemid vector pUC 119 and helper phage M13K07 were obtained from Dr. T. Freidman of Michigan State University. Plasmids pAPZ 71 (containing the complete apu gene) in E. coli SURE, and pAPZ 72 (containing a fusion construct of apu gene to the tac Z promoter in pUC 18) in E. coli DHSa, are the parental plasmids used in this work.'

Enzymes-Restriction enzymes were obtained from Bethesda Re- search Laboratories, United States Biochemical Co. (Cleveland, OH), or Boehringer Mannheim.

Ol~gonucleotides-Oligonucleotides were synthesized in an Applied Biosystems model 380A DNA synthesizer at the Macromolecular Facility, Department of Biochemistry, Michigan State University. Purification of the oligonucleotides used in site-directed mutagenesis was performed using thin layer chromatography (Sure Pure oligo- nucleotide purification kit; United States Biochemicals) and sub- sequently 5'-phosphorylated using T4 polynucleotide kinase, for construction of o l i~nucleo t ide-d i rec~ mutants (Oligonucleotide- directed in vitro Mutagenesis System Version 2.1, Amersham Corp.).

Enzyme Assays-For assay of pullulanase activity, 160 pl of 1.25% (w/v) pullulan (for pullulanase activity) in 50 mM acetate buffer, pH 6.0, containing 5 mM CaClz and 40 p1 of enzyme solution were mixed and incubated at 60 "C for 30 min. The reaction was stopped by adding 0.8 ml of dinitro salicylate solution (Miller, 1959) and heated in a boiling water bath for 15 min. The samples were cooled on ice and the absorbance of the reaction solution measured at 640 nm. One unit of pullulanase activity is defined as the amount of enzyme which produces 1 pmol of reducing sugar (with glucose as the standard)/ min under the assay conditions.

For assay of a-amylase activity, 160 pl of 1.25% (w/v) soluble starch in 50 mM acetate buffer (pH 6.0) containing 5 mM CaClZ and 40 pl of enzyme solution were mixed and incubated at 60 "C for 30 min. The reaction was stopped by adding 0.8 ml of dinitro salicylate solution and heated in a boiling water bath for 15 min. The samples were cooled on ice and the absorbance of the reaction solution measured at 640 nm. One unit of a-amylase activity is defined as the amount of enzyme that produces 1 pmol of glucose/min under the assay conditions described.

Protein D e t e r m ~ ~ ~ i o n and Gel Electrophoresis-Protein concen- trations were determined using the Bio-Rad protein assay kits based on the dye binding assay of Bradford (1976) or using bicinchon~ic acid (BCA Assay Kit, Pierce Chemical Co.), using bovine serum albumin as standard. SDS-PAGE was performed according to the method of Laemmli (1970) using 7.5% polyacrylamide gels in a Mini- Protean I1 apparatus (Bio-Rad), and protein bands were visualized by staining with Coomassie Brilliant Blue R-250. The molecular weights of the recombinant proteins were determined using high range molecular weight standards (Bio-Rad) containing myosin (200,000), @galactosidase (116,250), phosphorylase b (97,400), bovine serum albumin (66,200), and ovalbumin (42,700).

Preparation of E. coli Competent Cells and Transformation-E. coli cells were made competent by the method by Hanahan (1983), as described by Perbal (1988). Transformation of competent cells was carried out as follows: 1 pl of the ligation reaction (IO ng of DNA) was mixed with 20 pl of competent cells and incubated on ice for 30 min in 1.5-ml Eppendorf microcentrifuge tubes, The mixture was heat shocked for 40 s at 42-44 "C and placed on ice for 2 min. 80 pl of cold SOC medium containing 2% (w/v) bactotryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCI, 10 mM MgC12, 10 mM MgSOd, and 20 mM glucose (Hanahan, 1983) was added and the transformation mixture incubated at 37 "C for 1 h in a rotary shaker a t 225 rpm. Transformants harboring recombinant pUC 119 plasmids were selected by plating the transformation sample onto 2 X YT agar (Sambrook et al., 1989) containing 50 pg/ml ampicillin, 5-bromo-4- chloro-3-indolyl-~-D-thiogalactopyranoside and isopropyl-/%D-thio- galacto pyranoside as described by Rodriguez and Tait (1983).

Construction of Nested Deletion Mutants for Sequencing-pAPZ 72

DNA, which contained a 5.4-kbp apu gene fragment,' was linearized with XbaI and the 5.4-kbp fragment isolated from an agarose gel (0.8% w/v) by electroelution. The 5.4-khp DNA fragment was ligated into phagemid vector pUC119 linearized with XbaI and transformed into E. coli DH5aF' competent cells. Directional subclones of the insert within pUC119 were identified by restriction mapping using the unique Sal1 and SphI restriction enzyme sites (originating from the respective multicfoning sites on the pUCl8 donor plasmid and pUC119 recipient plasmid) within the recombinant pUC119 vector. Inserts in opposite orientations were denoted pAPZl18 and pAPZl19. The individual plasmids were double digested with SstI and BamHI and used to create nested sets of deletion mutants (Ausubel et al., 1988) (Fig. 1). Single-stranded DNA generated from the deletion mutants by superinfection of phagemid containing DH5aF' cells with helper phage M13K07 was used to deduce the DNA sequence of the amylopullulanase gene by the dideoxy chain termination method (Sanger et aZ., 1977). The regions external to the 5.4-kbp apu DNA fragment were sequenced by double-stranded DNA sequencing of pAPZ 71 plasmid DNA using synthetic oligonucleotides.

Production of Single-stranded DNA from Phagemid Vectors- Helper phage M13K07 was grown and purified as described previ- ously (Sambrook et al., 1989). For production of single-stranded DNA, a fresh bacterial colony (grown on 2 X YT agar containing 50 pg/ml ampicillin) harboring pUC 118 or pUC 119 recombinant phagemid containing the 5.4-kbp opu gene insert (from pAPZ 72) was suspended in a 15-ml culture tube (Corning, New York, NY) containing 3 mi of 2YT (Sambrook et al,, 1989) media. The medium was adjusted to 100 pg/ml with ampicillin and 30 pl of helper phage M13K07 (2 X lo9 plaque-forming units/ml) was added to a final concentration of 2 X lo7 plaque-forming units/ml. The tubes were incubated at 37 "C, at 250 rpm for 1.5 h, or until slightly turbid. Kanamycin was added to a final concentration of 70 pg/ml and incubation continued for a further 12-14 h more at 300 rpm, at 37 "C. The culture was centrifuged at

pAPZ72

" " - x - 5 e

FIG. 1. Nested deletion mutant constructs used for sequenc- ing the apu gene. The hatched area represents T. ethumlicus 39E DNA insert; mcs, multicloning site. The solid areas represent part of the T. ethanolicus 39E DNA insert after nested deletion mutations.

16334 Sequencing and Site-directed Mutagenesis of apu Gene 17,000 X g for 10 min at 4 "C and the supernatant recovered. The phagemid particles were precipitated by adding to the 3 ml of super- natant, 334 mi of 40% (w/v) PEG-8000 and 334 ml of 5 M sodium acetate (pH 7.0). The phagemid was allowed to precipitate on ice for 15 min and collected by centrifugation at 17,000 X g for 10 min. The phagemid pellet was resuspended in 0.3 ml T E buffer (10 mM Tris, 1 mM EDTA (pH 8.0)) and extracted three times with an equal volume of phenol/chloroform (3:l v/v) by gentle mixing for 5 min. After a final extraction with an equal volume of chloroform/isoamyl alcohol (241 v/v), the single-stranded DNA was precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volume of ethanol, by chilling at -70 "C for 30 min, and subsequent centri~gation for 30 min. The DNA was redissolved in 10 r l of water and used for DNA sequencing or site- directed mutagenesis.

DNA Sequencing-Sequenase V.2.0 DNA polymerase and Sequen- ase V.2.0 sequencing kit from United States Biochemicals were used for DNA sequencing. For single-stranded DNA sequencing, the pro- tocol as described by the manufacturer was used (Instruction manual, Sequenase V.2.0, United State Biochemicals). For double- strand^ DNA sequencing, denaturation of double-stranded plasmid DNA was performed as described by Zhang et al. (1988). For sequencing reac- tions where lac Z fusion constructs were involved (pAPZ 119), uni- versal M13/pUC forward sequencing primer was used. For double- stranded DNA sequencing, within the 5' EcoRI-HindIII region of the 6.1-kb DNA insert of the original recombinant plasmid pAPZ 71, synthetic oligonucleotides were used as primers.

Sequence Analysis-The amino acid sequence inferred from the amylopullulanase DNA sequence was compared with the primary structures of a-amylases and pullulanases available through GenBank (IntelliGenetics Inc., Mountain View, CA). GCG Sequence Analysis Software Package V.7.0 (Devereux et ut., 1984) was used in the analysis and multiple sequence alignment and subsequent data ma- nipulations. Hydrophobic cluster analysis (HCA) of the amino acid sequences was performed as described by Gaboriaud et at. (1987).

Template DNA pAPZ 76

P r i m e r Aap-Asn GZ 52

P r i m e r Asp-Glu GZ 69

Template DNA p A P Z 7 6

P r i m e r Glu-Gln GZ 56

Primer Glu-Asp GZ 88

Template DNA PAP2 76

P r i m e r Asp-Asn GZ 55

P r i m e r Asp-Glu GZ 68

~ l ~ o n ~ ~ o t ~ e - d i r e c t e d ~ u ~ e ~ s ~ of A m y ~ p u ~ u ~ n ~ e Gene- Oligonucleotide-directed in vitro mutagenesis system V.2.1 from Amersham Corp. was used in constructing single point base mutants (Nakamaye and Eckstein, 1986). E. coli TG-1 was transformed with a nested deletion mutant, containing an upu gene fragment of 2.9 kb, denoted pAPZ 12-3 (in pUC 119), which maintained both a-amylase and pullulanase activities, as well as the~ostability. This was used to generate single-stranded DNA, using M13K07 helper phage, for use as the template for oligonucleotide-directed mutagenesis. The synthetic oligonucleotide primers were designed to be complementary to the single-stranded template DNA and contained the appropriate single point base mismatches as shown in Fig. 2.

Following in vitro mutagenesis, plasmid DNA was isolated from E. coli TG-1 transformants which did not express amylopullulanase activity (Fig. 3). The 0.9-kbp KpnI-BglII DNA fragment was isolated from the plasmids by double digestion with KpnI and BglII, followed by agarose 1.2% (w/v) gel electrophoresis and electroblotting onto NA-45 membranes (Schleicher & Schuell). pAPZ75 plasmid DNA lacking the 0.9-kbp BglII-KpnI fragment was prepared by digestion with BglII and KpnI, followed by agarose gel electrophoresis and electroelution (Elutrap, Schleicher & Schuell). The individual 0.9- kbp DNA fragments recovered from the site-directed mutants were ligated to the linearized vector pAPZ75 lacking the 0.9-kbp DNA region, and transformed into E. coli TG-1. Single-stranded DNA was recovered from the transformants by infection with M13K07 helper phage as described previously. Nucleotide sequence of the single point base mutants were confirmed by sequencing the 0.9-kbp KpnI-BglII region using synthetic oligonucleotides.

Enzyme Purification-E. coli TG-1 harboring the pAPZ 12-3 re- combinant plasmids containing the specific single point base muta- tions were grown at 37 "C in 5.0 ml of LB media containing ampicillin (50 pg/ml) and isopropyl-8-D-thiogalactopyranoside (1 mM). Cells were harvested by centri~gation in a microcentri~ge (14,000 rpm X 1 min) and the cell pellet sonicated in 1.0 ml of 50 mM acetate buffer

597 5' C TGG AGA TTG GTT GCA AA- --- - 3'

Trp Arg Leu Asp Val Ala

3' G ACC TCT AAC ZTA CAA CGT TT- --- - 5'

3' - -CC TCT AAC CTX CAA CGT TTT AC- - 5'

5'

3'

3'

62 6 A ATG ATT GCA a CTT TGG GGA GA

Met Ile Ala Glu Leu Trp GLy 3'

T TAC TAA CGT GTT GAA ACC CC- -- 5'

- TAC TAA CGT CT& GAA ACC CCT CTA CGA 5'

703

Leu Gly Ser H i s Asp Thr Met Arq Ile 5' TTA GGT TCT CAT ACC ATG AGA ATA 3'

3' --T CCA AGA GTA XTG TGG TAC TC- "- 5'

3' "- -CA AGA GTA CTJ= TGG TAC TCT T" 5'

pAPZ 76 is identical to pAPZ 12-3 (in pUC 119) except for growth in E. coli TG-1. FIG. 2. Synthetic oligonucleotide primers used in oligonucleotide-directed mutagenesis of active site amino acids. Plasmid

Sequencing and Site-directed Mutagenesis of apu Gene 16335

- - " "

- 0 3 c c

pAPZ7 1

I .O 2.0 3.0 4.0 5.0 6.0 7.0 0.0 9.0

aP" " " -

5 pUC 119

W

lac z I pAPZ7 6

FIG. 3. Strategy for constructing oligonucleotide-directed mutants. The hatched area represents T. ethaml- Bglll/KPnl icus 39E DNA insert, apu represents the gene encoding amylopullulanase activ- - ity. "

V- - .c 5 I m

I Electroelute Site directed mutagenesis

- - - I .= t; E I am" v - -

Select negative mutant

lac Z k-1 pUC 119 pAPZ7 6 Bglll/Kpnl

"

X m z s

isolate 0 .9 kb fragment by elecroblotting

(pH 6.0) containing 5 mM CaC12. The cell lysate was centrifuged (14,000 rpm X 5 min) and the supernatant heat treated at 85 "C for 5 min. After centrifugation (14,000 rpm X 5 min), the supernatant was recovered and tested for a-amylase and pullulanase activity.

RESULTS

Subcloning and Construction of Nested Deletion Mutants- In order to deduce the primary sequence of the amylopullu- lanase by sequencing of the 6.1-kbp DNA insert containing the apu gene, nested deletion mutants were constructed from the 5' and 3' ends of the gene. The nested deletion mutants constructed from subclones pAPZ118 and pAPZ119 are shown in Fig. 1. Since the open reading frame of 5.3-kbp Hind1111 XbaI fragment of the apu gene is in the correct orientation to the lac Z promoter of the pUC119 vector, and in the correct reading frame, amylopullulanase was expressed by E. coli harboring the pAPZ119 or harboring mutants constructed by deletion from the 3' end of the gene. In pAPZ118, however, the gene was in the opposite orientation to the lac Z promoter, and therefore, the enzyme was not expressed.

Nucleotide Sequence of T. ethanolicus 39E apu Gene-In order to deduce the primary sequence of amylopullulanase, a 5-kbp region within the 6.1-kbp chromosomal DNA insert was sequenced (Fig. 4). The 5000-bp segment contained an open reading frame of 4443 bp, starting at nucleotides 331-

I ligate

Sequence Bglll/Kpnl fragment using synthetic oligonucleotides

333 with a GTG codon, and terminated at 4773 bp, followed by termination codons at 4774 bp (TGA) and at 4796 bp (TAA) positions. The reading frame deduced from the DNA sequence was confirmed by NH2-terminal amino acid se- quencing, which was identical to the first 7 amino acids of the native enzyme (Mathupala and Zeikus, 1993). The predicted primary sequence encoded by the open reading frame corre- sponds to 1450 amino acids with an estimated molecular weight of 162,780, which agrees closely with the M, of 160,000 obtained by SDS-PAGE analysis for the enzyme expressed in E. coli, although it is higher than the M, 140,000 determined for the native enzyme (Saha et al., 1988). The GTG initiation codon is preceded with a spacing of 5 bp, by a putative ribosomal binding site (5'-AAAGGGGG-3') exhibiting strong similarity to the 16 S rRNA of B. subtilis (McConnell et al., 1986). The structural gene is preceded by a putative promoter sequence, similar to the consensus promoter sequences of E. coli and B. subtilis (Rosenburg and Court, 1979; McConnell et al., 1986). A region (5'-TATAAT-3') similar to the -10 consensus promoter sequence of E. coli can be identified 158 bp upstream of the putative ribosome-binding site, preceded by a region (5'-TTGACA-3') similar to the -35 consensus promoter sequence of E. coli. Also recognizable upstream of these regions is a sequence resembling the consensus sequence for binding of the CAMP-CAP complex of E. coli (Crom-

16336 Sequencing and Site-directed Mutagenesis of apu Gene

FIG. 4. Nucleotide sequence and the deduced amino acid sequence of apu gene of T. ethanolicus 39E. CAMP, sequence resembling the consen- sus sequence for binding of CAMP-CAP complex of E. coli. RBS = putative ri- bosome binding site. -35 = sequence similar to -35 consensus promoter se- quence of E. coli. -10 = sequence similar

E. coli. The initial underlined deduced to -10 consensus promoter sequence of

amino acid sequence refers to the puta- tive signal sequence. Box represents the NH2-terminal amino acid sequence of the processed protein.

-330

-279

-228

-177

-126

-75

-24

28

79

130

181

232

283

334

385

436

487

530

589

640

TGTTCTTACTGTTTACAATACTTTTAGCA-CAGATAAGATGGAGA

TGTATTTATAAACACATTTATC-TTTATGAAAGCGAWGCCTA

T T A T T G T C A T T T C T C A G C A G T G T T T G G C T C A A T G C C T G X

CAATTTGCAAAAGCTGAGACAGATACGGCGCCTGCTATAGCCAATGTTGTT ~ 1 u T h r A s p T h r A l a P r o A l ~ l e A l a A s n V a l V a l

+1 10

GGCGATTWCAATCAAAGATTGGAGATTCTGACTGGAATATAAACAGTGAC GlyAspPheGlnSerLysIleGlyAspSerAspTrpAsnIleAsnSerAsp

20

AAAACAGTAATGACATATAAAGGTAATGGCTTTTATTTCCCCA LysThrValMetThrTyrLysGlyAsnGlyPheTyrGluPheThrThrPro 30 40

GTTGCGTTACCTGCAGGTGATTATGAGTATAAAG'LTGCT ValAlaLeuProAlaGlyAspTyrGluTyrLysValA1aLeuAsnHisSer

50 60

TGGGAAGGTGGAGGAGTTCCTTCACAAGGTAAmAAGCTTGT TrpGluGlyGlyGlyValProSerGlnGlyAsnLeuSerLeuH~sLeuAsp

70 80

TCAGATTCTGTAGTAACTTTTTATTACAACTATAATACTTCAAGTGTTACT SerAspSerValValThrPheTyrTyrAsnTyrAsnThrSerSerValThr

90

GATTCTACAAAATATACACCCGGA?iGAAAAACTTCCAAGhATTGTA AspSerThrLysTyrThrProIleProGluGluLysLeuProArgIleVal

100 110

GGTACTATACAATCGAGCAGGTGATGATTGGAAACCTGAM4CA GlyThrIleGlnSerAlaIleGlyAlaGlyAspAspTrpLysProGluThr

120 130

T C G A C A G C T A T A A T G A G A G A C T A T A A G ~ A A ~ ~ ~ A C ~ T A ~ C T SerThAlaIleMetArgAspTyrLysPheAsnAsnValTyrGluTy~h

140

G C A A A T G T T C G G T A T T A ~ ~ A A A G T A A C m A AlaAsnValProLysArgTyrTyrGluPheLysValThrLeuGlyProSer

150 160

n ; G G A T A T A T A A A T T A n ; G C T T A A A T G G ~ ~ T G G T C ~ T A ~ C T TrpAspIleAsnTyrGlyLeuAsnGlyGluGlnAsnGlyProAsnI~ePro

170 180

T I Y ; A A n ; T A G C C T A T G A T A C T A A G A T T A ~ ~ ~ T A ~ ~ G G ~ LeuAsnValAlaTyrAspThLysIleThrPheTyrTyrAspSerValSer

190

-280

-229

-178

-127

-76

-25

27

78

129

180

231

282

333

384

435

486

537

588

639

690

FIG. 4-continued

Sequencing and ~i t e -d i re~ ted ~ ~ t ~ e ~ s ~ of apu Gene

691

742

793

844

895

94 6

997

1048

1099

1150

1201

1252

1303

1354

1405

1456

1507

1558

1609

C A T A A T A T A ~ C A ~ T T A ~ T C C A C C T C P C A C A G G G HisAsnIleTrpThrAspTyrAsnProProLeuThrGlyPrOASpASnASn 200 210

ATATATTATGACGATTTA?UNCATGACACCCATGACCCATTCTTCCGCTTC IleTyrTyrAspAspLeuLysHisAspThrHisAspProPhePheArgPhe

220 230

G C T T T C G ( ; T G C T AlaPheGlyAlaIleLysThrGlyAspThrVa1Thr~uArgIkG~nAla

240 250

AAAAATCATGACCTCAGCTAAAATTTCITATTGGGATGATATTAAA LysAsnHi~spLeuGluSerAl~ysIleSerTyrT~AspAspIleLys

260

A A A A C A A G A A C A G A A G T C C C G A T G T A T ~ ~ ~ G ~ C ~ C ~ LysT~Ar~hrGluValPr~tTyrLysIleGlyGlnSerProAspGly

270 280

CAATATGAATA~TACn;GGAAGTGAAGTTATCCCA~~TT GlnTyrGluTyrTrpGluValLysLeuSerPheAspTyrProThrArgIle

290 300

TGGTATTACTTTATACTTAAAGACGGGACAAMACTGCTTATTACGGAGAT TrpTyrTyrPheIleLeuLysAspGlyThrLysThrAlaTyrTyrGlyAsp

310

A A C G A ~ T T A ~ ~ A ~ ~ ~ ~ ~ T A C G ( ; T A A A T A A A *

AsnAspGluGlnLeuGlyGlyValGlyLysAlaThrAspThrValAsnLys 320 330

GACTTTGAACTTACTGTATACGTATACGATAAAAA'ITTAGACACCCCTGATTGGATG AspPheGluLeuThrValTyrAspLysAsnLeuAspThrPraAspTrpMet

340 350

AAAGGGGCAGTAATGTATCAA?iTATTCCCAGATAGATTTTACAATfXl%AC LysGly~aVal~tTy~lnIlePhePr~~gPhe~rAsnGlyAsp

360

C ~ ~ ~ C C G C ~ ProLeuAsnAspArgLeuLysGluTyrSerArgGlyPheAspProValGlu 370 380

T A T C A ~ C G A C n ; G T A ~ C C ~ C C G A C A A T C C G A A TyrHisAspAspTrpTyrAspLeuPraAspAsnProAsnAspLysAspLys

390 400

CCTGGATATACAGGGGATGGTATATGAATAATGACTTC'ITEGTGGWT Prd;lyTyrThrGlyAspGlyfleTrpAsnAsnAgpPhePheGlyGlyAsp

410 420

TTACAAGGTATAAATGATAAATTGGATTATCTWCCTEGAATATCA LeuGlnGlyIleAsnAspLysLeuAspTyrLeuLysAsnLeuGlyIleSer

4 30

GfiATTTA~T~~CARmTCCAATCACCTTCCAATCACCGATA~T ValIleTyrLeuAsnProIlePheG1nSerProSerAsnHisArgTyrAsp

440 4 50

A ~ C C ~ T T A C A ~ ~ T A G A ~ ~ A T T G G G A G A ~ A G A T A C A T T T ThrThrAspTyrTPhrLysIleAspGluLeuLeuGlyAspLeuAspThrphe

460 470

AAAACACTTATGAAAGARGCCCA~GTATACGA~~AAAGTAATAC~T LysThrLeuMetLysGluAlaHlsAlaArgGlyIleLysValIleLeuAsp

480

GGCGTCTTCAATCATACAAGTG~TGATAGTATTTATTTTGATAGATAC~ GlyValPheAsnHisThrSerAspAspSerIleTyrPheAspArgTyrGly

490 500

A A G T A C T n ; G A T A A T G A A T P A G G n ; C T T A T C A A G C C T G G A L y s ~ r ~ u A s p A s n G l ~ ~ l y ~ a T y ~ l n A l a T ~ L y s G l n G l y A s p

510 520

16337

741

792

843

894

945

996

1047

1098

1149

1200

1251

1302

1353

1404

1455

1506

1557

1608

1659

16338 Sequencing and Site-directed Mutagenesis of apu Gene

FIG. &"ontinued

1660

1711

1762

1813

1864

1915

1966

2017

2068

2119

2170

2221

2272

2323

2374

2425

2476

2521

2578

CAGTCAAAATCTCCATACGGTGACTGGTACGAAATTAAGCCTGACGGTACC GlnSerLysSerPzo~rGlyAspTrp~rGluIleLysProAs~lyThr

530

TATGAGGGCTGCXGGGGATITGACAGCTTACCGGTAATAAGG TyrGluGlyTrpTrpGlyPheAspSerLet@roValIleArgGlnIleAsn 54 0 550

GWIRGTGAGTACAATGTAkAAAGTn;GGCAGATTTTATCRCT GlySerGluTyrAsnValLysSerTrpAlaAspPheIleIleAsnAsnPro

560 570

AATGCAATATCTAAGTATTGGTTAAATCCTGATGGGGATAAAGATGCAGGT AsnAlaIleSerLysTyrTrpLeuAsnProAspGlyAspLysAspAlaGly

580 590

GCAGATGGCXGAGATTGGATGTCTTCCAkAn;AAATKiCTC AlaAspGlyTrpArgLeuAspValAlaAsnGluIleAlaHisAspPheTrp

600

GTTCATTTTAGAGCTGCAATTAATACTGTGAAACCAAATGCGCCAATGATT ValHisPheArgAlaAlaIleAsnThrValLysProAs~laPr~tIle

610 620

G C R G A A C m G G ~ G A T G T T C ~ ~ C AlaGluLeuTrpGlyAspAlaSerLeuAspUuLeuGlyAspSerPheAsn

630 640

TCTGTTATGAACTATCT"TTTAGAAATGCAGTTATTG&"TTATACTCGAT SerValMetAsnTyrLeuPheArgAsnAlaValIleAspPheIleLeuAsp

650

AAACAGTTTGATGATGWTGTGGTTCACAATCCTATAGATGCAGCW LysGlnPheAspAspGlyAsnValValHisAsnProIleAspAlaAlaLys

660 670

CTTGACCAAAGGCTTATGAGCATATATGAGAGATATCCTCTTCCAGTATTT LeuAspGlnArgLeuMetSerIleTyrGluArgTyrProLeuProValPhe

680 690

TATTCTACTATGAACCTTTTAGGTTCTCATGACACCATGAGAATATTGACA qrSerThr~~snLeuLeuGlySerHisAspThr~tArg~leLeuThr

700

~ A ~ G G A T A T A A C T C ~ C T A A ~ ~ ~ T C ~ ~ G G C G A A A ValPheGlyTyrAsnSerAlaAsnGluAsnG1nAsnSerGlnGluAlaLys 710 720

GACCTTGCAGTTAACAGGCTTARACTTGCCGCAATATTGCAAATGGGCTAT AspLeuAlaValLysArgLeuLysLeuAlaAlaIleLeuGlnHetGlyTyr

730 740

CCGGGAATGCCTTCTATTTACTATGGTGACGAGGCAGGACAATCTGGTGGA ProGlyMetProSerIleTyrTyrGlyAspGluAlaGlyGlnSerGlyGly

750 760

AAAGACCCAGATAACAGCA~CTCTT~GA~GATAAAGAT LysAspPraAspAsnArgAr4ThrPheSerTrpGlyArgGSP

770

~ C A ~ ~ C ~ A A ~ G T C G T A A A C A T A A G G A A T G A A A A T LeuGl~spPhePheLysLysValValAsnIl~rgAsnGluAsnGlnVa~

780 790

T T R A A A R C A G G A G A C C T T G A R A C T T T A ~ ~ ~ G C G A T C LeuLysThrGlyAspLeuGluThrLeuTyrAlaAsnGlyAspValTyrAla

800 810

TTTGGAAGAAGAATTATAAATGGAAAAGATGTATTTGGTAATTCTTCCT PheGlyArgArgIleIleAsnGlyLysAspValPheGlyAsnSer~rPrO

820

GACAGTGTAGCTATTGTTGTGATTAATAAAGGTGAGGWGTCAGTAW AspSerVafAlaIleVa1ValIleAsnLysGfyGluAlaLysSerValG~n

830 840

1710

1761

1812

1863

1914

1965

2016

2067

2118

2169

2220

2211

2322

2373

2424

2475

2526

2577

2628

Sequencing and Site-directed Mutagenesis of apu Gene 16339

FIG. 4"continued

2629

2680

2731

2782

2833

2884

2935

2986

3037

3088

3139

3190

3241

3292

3343

3394

3445

3496

ATAGATACTACTAAATTTGTAAGAGATGGAGTTGCTTTTACAGATGCCTTA IleAspThrThrLysPheValArgAspGlyValAlaPheThrAspAlaLeu

850 860

AGTGGTAAGACATACACGGTTCGTGATGGACAAATTGTTGTAGAAGTTGTG SerGlyLysThrTyrThrValArgAspGlyGlnIleValValGluValVal

870

GCATTGGATGGGGCTATACTCATTTCAGATCCAGGACAGAATTTGACGGCA AlaLeuAspGlyAlaIleLeuIleSerAspProGlyGlnAsnLeuThrAla 880 890

CCTCAGCCAATAACAGACCTTAAAGCAGTTTCAGGAAATGGTCAAGTAGAC ProGlnProIleThrAspLeuLysAlaValSerGlyAsnGlyG1nValAsp

900 910

CTTTCGTGGAGTGCAGTAGATAGAGCAGTAAGTTATAACATTTACCGCTCT LeuSerTrpSerAlaValAspArgAlaValSerTyrAsnI1eTyrArgSer

920 930

ACAGTCAAAGGAGGGCTATATGAAAAAATAGCTTCAAATGTTACGCAAATT ThrValLysGlyGlyLeuTyrGluLysIleAlaSerAsnValThrGlnIle

940 .

ACTTATATTGATACAGATGTTACCAATGGTCTAAAGTATGTGTATTCTGTA ThrTyrIleAspThrAspValThrAsnGlyLeuLysTyrValTyrSerVal

950 960

ACGGCTGTAGATAGTGATGGAAATGAAAGTGCTTTAAGCAATGAAGTTGAG ThrAlaValAspSerAspGlyAsnGluSerAlaLeuSerAsnGluValGlu

970 980

GCATATCCAGCATTTTCTATTGGTTGGGCAGGAAATATGAACCAAGTTGAT AlaTyrProAlaPheSerIleGlyTrpAlaGlyAsnMetAalAsp

990

ACCCATGTAATAGGCGTAAATAATCCAGTTGAAGTTTATGCTGAAATTTGG ThrHisValIleGlyValAsnAsnProValGluValTyrAlaGluIleTrp

1000 1010

GCAGAAGGATTAACAGATAAACCTGGCCAAGGGGAMATATGATTGCCCAG AlaGluGlyLeuThrAspLysProGlyGlnGlyGluAsnMetIleAlaGln

1020 1030

TTAGGATATAGGTATATTGGAGATGGTGGGCAAGAn;CTA LeuGlyTyrArgTyrIleGlyAspGlyGlyGlnAspAlaThrArgAsnLys

1040

GTAGAAGGTGTTGAAATAAATAAGGACTGGACATGGGTTGATGCACGGTAT ValGluGlyValGluI1eAsnLysAspTrpThrTrpValAspAlaArqTyr 1050 1060

GTAGGGGATTCTGGAAATAACGACAAATACATGGCTAAATTTGTACCTGAT ValGlyAspSerGlyAsnAsnAspLysTyrMetAlaLysPheValProAsp

1070 1080

ATGGTAGGCACATGGGAATATATTATGAGATTTTCCTCTAACCAAGGACAG MetValGlyThrTrpGluTyrIleMetArgPheSerSerAsnGlnGlyGln

1090 1100

G A T T G G A C G T A T A C A A A A G G G C C A G A T G G G A A A A C A G A T G G AspTrpThrTyrThrLysGlyProAspGlyLysThrAspGluAlaLysGln

1110

TTTATTGTCGTGCCATCAAATGATGTAGAACCACCTACAGCTCTAGGCTTA PheIleValValProSerAsnAspValGluProProThrAlaLeuGlyLeu

1120 1130

CAACAACCAGGAATTGAATCCTCAAGAGTTACACTTAACTGGAGTCTCTCA GlnGlnPrOGlyIleGluSerSerArgValThrLeuAsnTrpSerLeuSer

1140 1150

2679

2730

2781

2832

2883

2934

2985

3036

3087

3138

3189

3240

3291

3342

3393

3444

3495

3546

16340 Sequencing and Site-directed Mutagenesis of apu Gene

FIG. 4"continued

3547

3598

3649

3700

3751

3802

3853

3904

3955

4006

4057

4108

4159

4210

4261

4312

4363

4414

44 65

ACTGATAATGTAGCTATCTATGGCTACGAAATATACAAATCTTTAAGTGAA ThrAspAsnValAlaIleTyrGlyTyrGluIleTyrLysSerLeuSerGlu

1160

ACAGGACCATTTGTAAAGATTGCAACTGTGGCTGACACTGTGTATAACTAC ThrGlyProPheValLysIleAlaThrValAlaAspThrValTyrAsnTyr

1170 1180

GTAGATACAGATGTAGTAAATGGAAAAGTGTACTATTATA ValAspThrAspValValAsnGlyLysValTyrTyrTyrLysValValAla

1190 1200

GTTGATACTTCTTTTAACAGAACAGCXTCAUTATAGTGAAAGCTACACCT ValAspThrSerPheAsnArgThrAlaSerAsnIleValLysAlaThrPro

1210

GATATAATACCTATCAAAGTGATATTTAATGTAACAGTCCCTGATTATACT AspIleIleProIleLysValIlePheAsnValThrValProAspTyrThr 1220 1230

CCTGATGACGGTGCAAATATTGCTGGAAACTTCCATGACGCTTTCTGGAAT ProAspAspGlyAlaAsnIleAlaGlyAsnPheHisAspAlaPheTrpAsn

1240 1250

CCAAGTGCCCATCAGATGACAAAGACAGGACCTAACACTTACAGTATTACA ProSerAlaHisGlnMetThrLysThrGlyProAsnThrTyrSerIleThr

1260 1270

TTGACTTTAAATGAAGGAACACAGCTTGAATATAAATATGCAAGGGGCAGC LeuThrLeuAsnGluGlyThrGlnLeuGluTyrLysTyrAlaArgGlySer

1280

TGGGATAAGGTAGAAAAAGGTGAATATGGAGAGGAAATTGCAAATAGAAAA TrpAspLysVa1GluLysGlyG1uTyrGlyGluGluIleAlaAsnArgLys

1290 1300

ATAACTGTTGTCAATCAAGGTTCAAATACCATGGTGGTAAATGACACAGTG IleThrValValAsnGlnGlySerAsnThrMetValValAs~spThrVal

1310 1320

CAAAGATGGAGAGACTTACCAATATACATTTATTCTCCAAAAGATAATACT GlnArgTrpArgAspLeuProIleTyrIleTyrSerProLysAspAsnThr

1330

ACAGTAGATGCAAATACAAACGAGATAGAGATTAAAGGCAATACCTATAAA ThrValAspAlaAsnThrAsnGluIleGluIleLysGlyAsnThr~rLys

1340 1350

GGTGCAAAAGTAACTATAAATGATGAATCTTTTGTACAACAAGAAAATGGC GlyAlaLysValThrIleAsnAspGluSerPheValGlnGlnGluAsnGly

1360 1370

GTATTTACAAAAGTAGTGCCTCTTGAATACGGTGTAAATACTACTAAAATA ValPheThrLysValValProLeuGluTyrGlyValAsnThrThrLysIle

1380

C A T G T G G A G C C G A G T G G T G A C A A G A A T A A T C A C A A HisValGluProSerGlyAspLysAsnAsnGluLeuThrLysAspIleThr 1390 1400

ATAACTGTTATAAGAGAGGAGCCTGTCCAGGAAAAAGAACCAACTCCTACG IleThrValIleArgGluGluProValGlnGluLysGluProThrProThr

1410 1420

CCAGAGTCTGAGCCAGCACCAATGCCTGAACCACAACCGACACCAACACCA ProGluSerGluProAlaProMetProGluProGlnProThrProThrPro

1430 1440

GAACCACAGCCTTCTGCAATTATGGCATTGTGACTGCCTCAACACTTAATT GluProGlnProSerAlaIleMetAlaLeu *

1450

TAAGAGAAGGAGCAAGTATCACAAGTMTTATAGGTACTATCTGCTGGG

3597

3648

3699

3750

3801

3852

3903

3954

4005

4056

4107

4158

4209

4260

4311

4362

4413

4464

4515

Sequencing and Site-directed Mutagenesis of apu Gene 16341

FIG. 4-continued

Taa 094 39E 465

Stpuyl 093 Cloepl 138

Bazamyla 078

393 582 TIa 190

Cloeml 241

Bacamyla 218 Stpuyl 191

Taa 220 Cloeml 270

Bacamyla 248

39E 611

stnuply1 220

Taa 289 393 685

Stmaryl 290 ClOeml 348

Bacamyla 324

4516

4567

4618

4669

I

YGTADDLKALSSALHERGMYLMVDWEM LGDLDTFKTLMKEAHARGIKVILDGVFNET

FGSFTDFQNLINTAHAHNIKVIIDFAPNET LGDRAAFKSMVDTCHAAGVKWADSVINEM YGTKAQYLQAIQAAHAAGMQVYADVVFDHK

NPDG. D K D A G ~ E I ~ D F ~ H F WVGSLVSNYSSDGLRIDTVKHVQKDFWPGY AIKVWLD.MGIDGIRLDAVKHMPFGWQKNF

W G K W Y V N T T N I D G E H I K F S F F P D W YLNDLLS.LGVDGFRIDAAKHMPAA.DLTA

11.

111 RAAINTVKPNAPMI~LWGDASLD. . . . . . MDSILSYRP.VFTFGEiiFLGTNEIDVNNTY NKA. ... A G . V Y C 1 ~ D P A Y T C P Y Q N

SYVRSQTGKPLFTVGEYWSYDINKLHNYIT 1KAKVGNGS.TYWKQEhIHGAGEAVQPSEY

E R Y P L P V F Y S T ~ ~ S ~ T M R I L T ~ G Y N IV

.......... LGTEVENHDNPRFASYTNDI

.......... SAVFVDNHDTER ... GGDTL . . . . . . . . . . MVTFIDNXDMDRFYNGGSTR

........... VTFVDNHDTNPAKRCSHGR

AAAGTTGTAAAATGGCTTGAGGAAGTGAATGGATGGTACAAAGTTGACTAT 4566

~ C G G C ~ G T A G G A T A T G ~ C A A C ~ T A T G ~ C ~ ~ A G T G C ~ ~ T 4617

CCATCAAAGGTRACCGTTGCARAATCAGTGAAAGTTATAGTGAAGAGCGGA 4668

TT

4 94 123

122 167

107

610 219 269 219 247

634

299 244

249 278

714 308 367 306 342

FIG. 5. Multiple sequence alignment of deduced amino acid sequence of upu from T. e t h a n o ~ i c ~ 39E with sequences of n- amylases using “Pileup” computer program (Devereux et d., 1984). The legend at the bottom of the figure identifies the organisms from which the sequences are represented. The four regions indicated by Roman numerals represent sequence motifs identified by align- ment with the four conserved regions of a-amylase. Putative catalytic residues targeted for site-directed mutagenesis on amylopullulanase are represented by closed circles. The dots represent regions within the sequences where an overall alignment could not be found.

brugghe et al., 1984), with the sequence 5’-TGTGC-3’. The NHAerminal of the deduced amino acid sequence contained a 31-amino-acid sequence motif putatively involved in cell- surface localization of the enzyme in T. ethanolicus 39E. Hydropathy profile analysis (data not shown) indicated the putative signal sequence as the most hydrophobic region within the deduced peptide sequence. The 31-amino-acid se- quence had strong similarity to typical secretion signal se- quences (McConnell et al., 1986).

Sequence Analysis-In order to identify the Conserved re- gions, the deduced sequence was aligned with known amino acid sequences of a-amylases and debranching enzymes, avail- able through GENBANK (Intelligenetics, Inc., Mountain View, CA). Amino acid sequence comparisons of the conserved regions between the deduced sequence of amylopullulanase with the sequence of enzymes capable of hydrolyzing a-1,4 bonds in polysaccharides are shown in Fig. 5. Amino acid sequence comparisons of the deduced sequence of amylopul- lulanase with conserved regions within the sequence of en- zymes hydrolyzing cu-1,6 bonds in polysaccharides are pre- sented in Fig. 6. It can be seen that the sequence of amylo- pullulanase contains four motifs, DGVFNH, DGWRLDVA, AELWG, LLGSHD, which are aligned with the consensus sequence motifs of enzymes capable of a-1,4 or a-1,6 glucos- idic bond hydrolysis. Previous studies (Mathupala and Zeikus,

4670

39t 473 mo 228

Al6glu 084 1s- 304

K8epul8 610

39t 585

Rl6plu 192 We0 314

I S O U 387 rt.epul8 686

39E 615 me0 346

1s- 415 Al6gl.u 241

K8epulr 718

39L 686

Al6plu 312 W w 407

1.08s 515 rt.epu1i 809

I LNKEA.HARGIKVILDFOTWBTSDDSIYFD LIDRC.HEKGXRVMLDA~C. ....... ~ Q ~ . H N A G I ~ ~ ~ T ~ G G T W T S LLHEM.HERNNKLMMDLVVMETSDEHNWF1

M I Q A I K Q D L G M N V I M T N A A G P . T D

G..DXDAGADOWRLLWANEIA......... TYWIREFDIDGWRLDWEID. . . . . . ... ..... EKGIDUFWDVINE’ISKEEGLPTVE AYWANTMGVDOlWDWSVLGN. ... SCLN A ~ T T D Y K I ~ ~ Y ~ ~ ~ I L S A ~

11

NT. ..VKPNAPMIAILLUGDASLDLLGDSFN ~ . . . L K P D ~ I L ~ ~ W L R G D Q F D .... LSHYDIMTVOPa?GVTTEEAKLYTGE ... ~ S A P N ~ P N ~ ~ S ~ A I N R I L ... KALNPDIYEF(nGWDSNQSD..REFIA

111

RYPLPVFYSTMN..LIGSZDTMRILTVFGY N

K W Q ~ E H T G W N S L ~ Q P R W S ~ G N SYPNNVNEAAFN..LIGSBDTSRILTVCGG

LFQSSGRSPWNSIMIDVEDGMTLKDVYSC LGMAGN.. .LADFVLXDX.DGAVKRGSEID

501

113 332 638

248

334 603

215

715 412

64 1 372 272 441 142

713 434 341 544 834

FIG. 6. Multiple sequence alignment of the deduced amino acid sequence of apu with sequences of a-1.6 hydrolyzing enzymes using the “Pileup” computer program (Devereux et d., 1984). The legend at the bottom of the figure identifies the organisms from which the sequences are represented. The four regions indicated by Roman numerals represent sequence motifs identified by alignment with the four conserved regions of a-amylases. Putative catalytic residues are represented by closed circles. The dots represent regions within the individual sequences where an overall alignment could not be found.

1993) using group-specific chemical m ~ f i c a t i o n of amylo- pullulanase had indicated the involvement of acidic amino acids in catalysis.

Identification and Modification of the Putative Catalytic Residues on A r n y ~ ~ ~ l L u l a n ~ e - F o r tentative identification of the catalytic residues of amylopullulanase, the amino acid sequence was compared with related enzymes previously in- vestigated with respect to their catalytic residues. The cata- lytic residues of Aspergillus oryzae a-amylase (taka-a-amy- lase, TAA) were reported to be either GluZ3’ in the third conserved region, and Aspzg7 in the fourth conserved region (Matsuura et al., 1984), or Aspzo6 in the second conserved region and Aspzg7 in the fourth conserved region (Buisson et al., 1987). Two conserved Asp (from the second and fourth regions) and one conserved Glu (from the third region) were proposed as the catalytic residues for neopuilu~anase from Bacillus stearotherrnophilus (Kuriki et al., 1991).

Hydrophobic cluster analysis revealed an apparent similar- ity among the hydrophobic cluster architectures of the amy- lopullulanase from T. ethunolicus 39E, TAA, and neopullulan- ase within the whole sequences from region I to IV. Moreover, 2 conserved Asp and 1 conserved Glu proposed as the catalytic residues of TAA and neopullulanase are located within well conserved segments of the HCA plots of all of the compared enzymes (Fig. 7). Since catalytic centers are usually highly

16342

39E

Taa

Neo

Sequencing and Site-directed Mutagenesis of apu Gene

FIG. 7. HCA plots of amylopullulanase (39E), taka-a-amylase (Tau), and neopullulanase (Neo). Numbering starts from the first amino acid of the mature protein for Taa and Neo. The first amino acid of the plot for 39E, R, is the 481st amino acid of the mature protein. Proline is symbolized by +, glycine by e, serine by El, and threonine by 0. Proposed catalytic residues are ringed with circles. For the parts of the sequences containing catalytic residues, the correspondences between hydrophobic clusters (segments 1-6) are shown by vertical lines.

TABLE I Activity of oligonucleotide-directed mutant constructs of apu gene Pullulanase activity is shown. In all mutants, concomitant loss of

a-amylase activity was also observed. Enzyme Mutation Specific activity'

unitlmg protein pAPZ75 None 53 (100%) D597N Asp6"-Asn 0.076 (0.14%) D597E Asp697-G1U 0.010 (0.19%) E6266 Glusm-Gln 0.050 (0.10%) E626D Gluam-Asp 0.076 (0.14%) D703N Asp7"-Asn 0.063 (0.12%) D703E Asp'"-Glu 0.050 (0.10%)

' Specific activity represents units of activity/mg protein. One unit of activity is defined as the amount of enzyme required to produce 1 pmol of glucose/min at pH 6.0 and 60 "C.

conserved during evolution, we suggest that Asp'", G1us2', and Asp703 of amylopullulanase from T. ethunolicus 39E may participate in catalysis, and therefore, these amino acids were targeted for site-directed mutagenesis studies to confirm their involvement in catalysis.

Mutagenesis of targeted residues resulted in both a-amylase and pullulanase activities being lost almost completely, indi- cating that Asp6" of amylopullulanase, corresponding to Aspzm of TAA in the second conserved region, Gluaz6 corre- sponding to the G1uZm in the third conserved region of TAA, and Asp703 corresponding to Aspm of the fourth conserved region of TAA, are tentatively involved in catalysis (Table I). To verify that the mutant proteins were fully processed and

1 2 3 4 5 7

' 200

t 116 ' 97

' 6 6

L 44

FIG. 8. SDS-PAGE of heat-treated extracts of E. coli car- rying the amylopullulanase gene (pAPZ7S) containing site- directed mutations. Lane I, E626D (GIufiZfi to Asp); lane 2, D703E (Asp703 to Glu); lane 3, native enzyme; lane 4, vector without insert; lane 5, standards. Amylopullulanase is indicated by the arrow on the left. The molecular weight (X of standards is indicated on the right.

thermostable, cell lysates of E. coli were subjected to heat treatment and examined by SDS-PAGE, which revealed the presence of a protein with an M, which was identical to the native enzyme (Fig. 8).

Alignment of the sequence of amylopullulanase with those of a-amylases, pullulanases, and glucoamylases is shown in Fig. 9. The apu sequence of T. ethumlicus 39E displayed similarities to a-amylase-pullulanase of C. thermhydrosul- furicum El01 (82%), a-amylase of A. oryzae (48%), neopul- lulanase of B. stearothermphilw (60%), and pullulanases of

Sequencing and Site-directed Mutagenesis of apu Gene

0 200 400 600 800 lo00 1 200 1400 I I I t 1 1 I I I I I I I I 1 1.0.

39E unylopuiluianasc

El01 a-amylase- -pullulanasr

K . w pullulmasr

K p n pullulmasr

16343

FIG. 9. Overall alignment of the deduced sequence of amylopullulanase of T. ethano~icue 39E with amylases from microbial and fungal origin. 39E, T. etharwlicus 39E E 101, C. ~~rmohydrosulfur~cum E 101; TAA, A. oryzae; B.amy, Bacillus amyloliquefaciens; K. ae, Klebsiella aerogenes; K. pn, K. pneumonia?. The open boxes represent regions putatively identified on all sequences based on the four conserved regions of a-amylase of A. oryzm.

Klebsiella spp. (44%). However, the overall similarity toward glucoamylases of Aspergillus spp. was much less, with a simi- larity of 40%.

It can be seen that the four conserved regions of all. the amylases are located in the center of the encoded peptide. It is interesting to note that the M, 100,000 peptide encoded by the 2.9-kbp nested deletion mutant constructed from the 4.4- kbp apu gene is located toward the center of amylopullulanase primary sequence, in alignment with the conserved sequence motifs of other amylases.

DISCUSSION

This is the first report demonstrating categorically that a single active site is responsible for the dual activities of amylopullulanase, an enzyme capable of hydrolyzing both a- 1,6 and a-1,4 bonds of polysaccharides. In addition evidence is provided for the possible involvement of 3 amino acids in Catalysis, from site-directed mutagenesis studies. Multiple sequence alignment of the deduced sequence of T. ethunolicus 39E showed greatest similarity toward a-amylase-pullulanase of C. t~rmohydrosulfur~cum El01 (82% similarity) and pul- lulanase of C. thermosulfurogenes (69% similarity). Analysis of the 5’ region of apu gene enabled the identification of a sequence motif for binding of the CAMP-CAP complex of E. coli (Crombrugghe et al., 1984) for regulation of gene expres- sion, which may be involved in catabolite (glucose) repression of pullulanase activity, reported previously for T. ethunolicus 39E (Hyun and Zeikus, 1985). Preliminary studies on the promoter region of a-amylase from A. oryzae had indicated the presence of an approximately 90-bp region 377-250 bp upstream to the a-amylase gene, which was necessary for expression and regulation of the a-amylase gene (Tada et al., 1991).

In order to clarify the true nature of catalysis, and to locate the apu gene region encoding the catalytic domain, we pro- ceeded to restrict the gene from the NHz-terminal end and the COOH-terminal end, testing each nested deletion mutant quantitatively for both a-amylase and pullulanase activities, as well as for thermostability. The smallest gene fragment, capable of expressing both activities and thermostability, was 2.9 kbp in length and contained sequence motifs similar to each of the four conserved regions identified on a-amylases, indicating that only one active site is present on apu gene. The 2.9-kbp gene fragment was identified within the apu gene by sequencing the 5’ and 3’ regions of the nested deletion mutant. This region corresponded to a polypeptide containing 965 amino acid residues, which was encoded by a region 2865 bp in length.

In the present study, HCA analysis of amylopullulanase

from T. ethunolicus 39E, a-amylase from A. oryzae, and neo- pullulanase from B. stearothermophilus was used to identify the location of the catalytic residues in amylopullulanase. AspSg7 and Asp703 of amylopullulanase were changed to Amsg7 and Asn703, respectively, by single point base mutations of the apu gene, and G W 6 was changed to Glnsz6. Complete loss of a-1,6 and a-1,4 cleavage activity was detected when all 3 residues were mutated individually. This result corresponds to the data concerning catalytic residues of the other glyco- sidases. Three acidic residues were proposed as the catalytic residues for neopullulanase from B. stearothermophilus (Ku- riki et al., 1991), cyclodextrin glucanotransferases from alkal- ophilic Bacillus sp. (Nakamura et al., 19921, and Bacillus circuluns (Klein and Schultz, 19911, a-amylase from B. stem rothermophilus (Holm et ai., 1990), cyclodextrinase from T. ethunolicus 39E (Podkovyrov et al., 1993) and endoglucanase from C. thermocellum (Chauvaux et al., 1992). For a-amylase from A. oryzae 1 Asp and 1 Glu, and for a-amylase from porcine pancrease 2 Asp were reported to be the catalytic residues, although these enzymes have the same (pa),, barrel super-secondary structure (Matsuura et al., 1984; Buisson et al., 1987). Site-directed mutagenesis studies of these a-amy- lases have not been reported to date, but a recent investigation of a-amylase from B. stearotherrnophilus by random mutagen- esis revealed that all 3 acidic residues are important for catalysis (Holm et al., 1990). Therefore, it is likely that 2 Asp and 1 Glu of a-amylases from A. oryzue and porcine pancrease may be involved in catalysis.

Since HCA plots of the regions bearing catalytic residues are similar in a-amylase from A. oryzue and amylopullulanase from T. ethanolicus 39E, we suggested that these enzymes are likely to have similar stereometry of the active site. Thereby, cleavage of glycosidic bonds by amylopullulanase may proceed through a catalysis mechanism analogous to that of at-amylase from A. oryzae (Matsuura et at., 1984). In this case, Asp703 of amylopullulanase which is in the hydrophilic environment may play the role of a general base. Both Aspss7 and G1P6 are in a non-polar environment, and at present it is unknown which of these adjacent residues is a proton donor. The second residue of this couple (either Aspsg7 or G1usz6) may promote donation of a proton by the first one by stabilizing the ionized carboxylate through hydrogen bonding, or by enhancing the ionization state (pK,,) of the residue directly involved in catalysis. The residues constituting the catalytic triad of amylopullulanase, Aspsg7, G1u626, and Asp703, are located within close proximity to each other, forming a single active site for the dual activities, in contrast to the dual active sites proposed for the a-amylase-pullulanase of C. thermohydro- sulfuricum El01 (Melasniemi et al., 1990) and amylase-pui-

16344 Sequencing and Site-directed Mutagenesis of apu Gene

lulanase of B. circulans F-2 (Sata et al., 1989). The loss of activity upon change of Aspsg7 to GluSg7, Asp703

to Glu703, or G1ufiZ6 to Aspfim indicates that the geometric alignment of the catalytic residues within the active center of amylopullulana~ is critical, in order to bring about catalysis. This contrasts with the flexibility shown by amylopullulanase toward a wide range of oligosaccharides and polysaccharides containing a-1,6 and a-1,4 linkages, which may be related to flexibility of the substrate binding residues rather than of the catalytic residues.

Amylopullulanase of T. ethanolicus 39E is an enzyme with greater flexibility toward a wide variety of polymeric sub- strates, in contrast to the amylases reported from other pro- karyotic or eukaryotic organisms, which are highly specific in catalysis toward either a-1,4 or a-1,6 linkages. Amylopullu- lanase may be considered as a nonspecific amylase which has the ability to cleave both a-1,4 and a-1,6 linkages, rather than as an a-amylase or a pullulanase which had acquired the ability to hydrolyze a-1,6 or a-1,4 bonds, respectively. Since amylopullulanase was isolated from a thermophilic anaerobe, which are believed to be among the first microorganisms to evolve on earth, the broad substrate specificity of this enzyme suggests that a-amylases and pullulanases may have evolved from an amylopullulanase type precursor enzyme. Therefore, this enzyme may be of great value in protein engineering studies to alter the substrate or product specificity of amy- lases, by changing the substrate binding or catalytic residues.

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