Novel Oxidatively Stable Subtilisin-like Serine ProteasesfS

KS*a

R

aodhwpsCzAafees

p

pMsbitlasgMbat

7

Biochemical and Biophysical Research Communications 279, 313–319 (2000)

doi:10.1006/bbrc.2000.3931, available online at http://www.idealibrary.com on

rom Alkaliphilic Bacillus spp.: Enzymatic Properties,equences, and Evolutionary Relationships

atsuhisa Saeki,* Mitsuyoshi Okuda,* Yuji Hatada,* Tohru Kobayashi,*usumu Ito,* Hideto Takami,† and Koki Horikoshi†,1

Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan;nd †Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237-0061, Japan

eceived November 10, 2000

overall sequence similarity between true subtilisinsaldhatAtaa

lhcesaiwbMiomracppu

athcEs

The genes for five subtilisin-like serine proteases fromlkaliphilic strains of Bacillus exhibiting resistance toxidative inactivation were cloned and sequenced. Theeduced amino acid sequences of the enzymes wereighly homologous (greater than 88% identity). Theyere composed of 638 or 639 amino acids, including aossible ;200-amino acid prepro-peptide, and uniquetretches of ;160 amino acids were found in the-terminal regions. The molecular masses of mature en-ymes (433 or 434 amino acids) were ;45 kDa for all.mino acid sequence comparison and phylogeneticnalysis indicated that these enzymes are far removedrom other known subtilisins in the line of molecularvolution. We propose that these novel proteases be cat-gorized as a new class of subtilisins, named oxidativelytable, alkaline protease. © 2000 Academic Press

Key Words: subtilisin; alkaliphile; oxidatively stablerotease; phylogenetic tree; evolution.

Subtilisin is one of the most studied bacterial serineroteases because of its industrial importance (1).ore than 200 proteases designated subtilases, the

uperfamily of subtilisin-like serine proteases, haveeen reported, and subtilases are subdivided into fam-lies A through F. Family A includes three groups, i.e.,rue subtilisins, high-alkaline proteases, and intracel-ular proteases (2). Horikoshi found the first high-lkaline protease (H-221), from alkaliphilic Bacillusp. strain no. 221 (3). The high-alkaline proteaseroup, including Savinase (4), PB92 (5), and-protease (6) that are all produced by alkaliphilic

acilli, has a low-molecular-mass of 26–28 kDa. Theyre used mainly in the detergent industry. Their ter-iary structures have been well solved (4, 7, 8). The

1 To whom correspondence should be addressed. Fax: 81-3-5765-102. E-mail: horikok@jamstec.go.jp.

313

nd high-alkaline proteases is relatively high, but phy-ogenetic analysis revealed that ;40 amino acid resi-ues of the former are replaced in the C-terminalalves of the molecule, possibly during adaptation tolkaline environments (9). In the crystal of M-protease,he molecule has a salt bridge triad comprising Arg19,rg265, and Glu269. Such a salt bridge is characteris-

ic of the structures of high-alkaline proteases, but isbsent from less alkaline true subtilisins Carlsbergnd BPN9 (9).To cope with extremes of alkaline pH and high che-

ator concentration in detergents, protein engineeringas been applied extensively to the subtilisin genes toreate subtilisins with greater thermal stability, tonhance resistance to chelators, to change substratepecificity, and to alter the pH- and temperature-ctivity profiles of the enzymes (10–12). However, ox-dative inactivation of subtilisins is an Achilles’ heelhen the enzymes are included in modern bleach-ased detergent formulations. Subtilisins all contain aet residue, located next to the catalytic Ser, that is

mplicated as a primary site for oxidative inactivationf the enzymes (13, 14). Although oxidatively stableutant enzymes are obtained by replacing the Met

esidue with a nonoxidizable amino acid, the aminocid replacement at this position extremely reducesatalytic power on protein substrates (15). Thus, therimary goals for an optimally performing detergentrotease are the high activity and oxidative stabilitynder alkaline pH conditions.In a 1974 Japanese patent (JP 740710), Horikoshi

nd Yoshida reported that a subtilisin-like serine pro-ease (E-1) from alkaliphilic Bacillus sp. strain D-6 wasighly resistant to the oxidant sodium perborate. Re-ently, oxidatively stable serine proteases similar to-1 have been also isolated from alkaliphilic Bacillustrains and documented in patents. Here we report

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

detailed enzymatic properties and cloning and se-qMzbpt

M

amaoyatFpF1F

E(sfwtd2vDbaTmtwoatosPw

wBl

BPtStCD

glidc

in the kit and oligonucleotide primers (A through F) designed for theaPcpgawCpGNLbiTEGrtwsoapfw1OsP

sPtcsSaapsswt

si1tattcCeoobauopAo

B0

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

uencing of the genes for E-1 and the patent enzymes.oreover, we show that these oxidatively stable en-

ymes belong to a new class of the subtilisin familyased on the results of phylogenetic analysis and areromising for use in bleach-based detergent formula-ions.

ATERIALS AND METHODS

Bacterial strains, plasmids, and propagation. Our stock culturesnd patent strains were grown on an alkaline agar that contained skimilk, as described previously (16). Colonies that formed halos on the

gar were picked up and grown in a liquid medium (pH 9.0) composedf 0.5% glucose, 0.2% Polypepton S (Nippon Pharmaceutical), 0.05%east extract (Difco), 0.1% KH2PO4, and 0.26% Na2CO3 (separatelyutoclaved). The oxidative stability against H2O2 of protease activity inhe centrifugal supernatant was examined by the method described inig. 1. The sources of the genes and enzymes used in this study wereatent strains of alkaliphilic Bacillus, FERM P-1592 (strain D-6),ERM BP-6534 (strain KSM-KP9860; PCT JP9804528), FERM BP-029 (strain Y; EP 204342 A2), NCIB 12289 (WO 8801293 A1), andERM P-11162 (strain SD521; JP 91191781 A2).

Purification of enzymes. The five Bacillus proteases, designated-1 (from strain D-6), KP-9860 (from strain KSM-KP9860), NP-1

from strain NCIB 12289), LP-Ya (from strain Y), and SD-521 (fromtrain SD521), were purified individually to homogeneity by theollowing procedure. Each centrifugal supernatant of the culturesas treated with ammonium sulfate, and the fraction that precipi-

ated at 90% saturation was collected. The precipitate formed wasissolved in a small volume of 10 mM Tris–HCl buffer (pH 7.5) plusmM CaCl2, and the solution was dialyzed overnight against a largeolume of the same buffer. The retentate was applied to a column ofEAE Bio-GelA (Bio-Rad) that had been equilibrated with the sameuffer. The column was washed with the buffer, and non-adsorbedctive fractions were combined and concentrated by ultrafiltration.he concentrate was dialyzed overnight against a large volume of 10M Tris-HCl buffer (pH 7.5) plus 2 mM CaCl2. The retentate was

hen put on a column of SP-Toyopearl 550W (Tosoh) equilibratedith the same buffer, and the protease was eluted using a gradientf 0–50 mM NaCl in the buffer. The active fractions were collectednd concentrated by ultrafiltration, and the concentrate was used ashe final preparation of purified enzyme. The purified enzymes thusbtained with yields of 15–20% were all homogeneous as judged byodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–AGE). A commercial high-alkaline protease, KAP (M-protease),as purified to homogeneity, as described previously (6).

N-terminal sequencing. The amino acid sequences of proteasesere each determined in a protein sequencer (model 476A; Appliediosystems) that was connected to an on-line PTH-derivative ana-

yzer.

Isolation of DNA and transformation. Genomic DNAs of the fiveacillus strains were prepared as described by Saito and Miura (17).reparation of plasmid DNA, restriction digestion, ligation, andransformation of Escherichia coli cells were done by the method ofambrook et al. (18). Sequencing was performed by the dideoxy chainermination method using a DNA sequencing kit (Dye Terminatorycle Sequencing Ready Reaction; Perkin-Elmer) and an automatedNA sequencer (model 377; Applied Biosystems).

Amplification and sequencing of DNA. The complete proteaseenes and their flanking regions were sequenced by the cassette-igation-mediated PCR method with a Takara LA PCR in vitro Clon-ng kit, according to the manufacturer’s instructions. Genomic DNAigested with EcoRI or PstI was ligated with the oligonucleotideassette having EcoRI or PstI cohesive ends. The C1 and C2 primers

314

mplification of appropriate genomic regions were used for PCR. TheCR products were purified with a High Pure PCR Product Purifi-ation kit (Boehringer Mannheim). The gene encoding N-terminalart of E-1 was first amplified by PCR from a EcoRI cassette-ligatedenomic DNA as template with primers C1 and A. The PCR-mplified DNA was used as template for the second round of PCRith primers C2 and B. Primer A, 59-AA(A/T/G/C)GA(T/C)GT(A/T/G/)GC(A/T/G/C)(A/C)G(A/T/G/C)GG(A/T/G/C)AT(A/T/C)G-39, andrimer B, 59-GC(A/T/G/C)GA(T/C)GT(A/T/G/C)GC(A/T/G/C)CA(A/)AA(T/C)AA(T/C)TA-39, were designed from the sequence of the-terminal end of mature E-1, Asn-Asp-Val-Ala-Arg-Gly-Ile-Val-ys-Ala-Asp-Val-Ala-Gln (see Fig. 2). The amplified fragment of 360p was sequenced directly. To determine the complete sequence,ncluding 59 and 39 regions, primers C (59-CCCACGGAATGC-TCATGCATAG-39), D (59-GTTCTTCCTAACGCGTAAAGAGC-39),

(59-GTAGCTGGTTCTGTGCTTGGTAATG-39), and F (59-AAA-GAATGGCTCCGCAAGCTAAC-39) were synthesized based on the

esults of sequencing of the fragment. The 59 region of the gene washen amplified by PCR from an EcoRI cassette-ligated genomic DNAith primers C1 and D. The PCR-amplified DNA was used for the

econd round of PCR with primers C2 and C. The amplified productf 2.5 kb was then sequenced directly. The 39 region of the gene wasmplified from a PstI cassette-ligated genomic DNA as template andrimers C1 and E. The PCR-amplified product was used as templateor the successive PCR with primers C2 and F. PCR experimentsere performed with 30 cycles of a 1-min denaturing step at 94°C, a-min annealing step at 50°C, and a 3-min extension step at 72°C.ther related protease genes presented in this study were cloned and

equenced in a similar manner by the cassette-ligation-mediatedCR method.

Construction of a phylogenetic tree. An extensive search of thecientific literature (PubMed: http http://www.ncbi.nlm.nih.gov/ubMed/) and databases (nr-aa, PIR, and Swiss-Prot) was performedo collect the amino acid sequences of enzymologically well-haracterized subtilisins using the Blast2 program (19). Amino acidequences of the 29 enzymes chosen, including E-1, KP-9860, LP-Ya,D-521, and NP-1, were aligned using the Clustal multiple-lignment program (Clustal X) (20). Sequence segments around cat-lytic residues (Asp32, His64, and Ser221 in BPN9 numbering) wereicked up and aligned to modify initial automatic alignment of allequences, because the fiver proteases sequenced in this studyhowed very low similarity to other enzymes. Sites involving gapsere excluded from all analyses. A phylogenetic tree was inferred by

he neighbor-joining method (21) in the Clustal X program ver.1.64b.

Other analytical methods. Protease activity was routinely as-ayed as follows. A total of 0.9 ml 0.67% Harmerstein casein (Merck)n a 50 mM buffer-0.1 ml enzyme solution was incubated at 35°C for0 min. The reaction was stopped by addition of 2.0 ml of 0.11 Mrichloroacetic acid/0.33 M acetic acid/0.22 M sodium acetate. Afterllowing the reaction mixture to stand for 30 min at room tempera-ure, the mixture was passed through filter paper (Whatman No. 2)o remove denatured proteins. The filtrate was used to determineaseinolytic activity by color reaction with the phenol reagent (Kantohemical). One unit of the activity was defined as the amount ofnzyme that produced acid-soluble peptides equivalent to one mmolf L-Tyr per min. Protease activity was also monitored with 5 mMligopeptidyl p-nitroanilide (pNA; Sigma) at pH 10.5 in 50 mMorate buffer. One unit of enzymatic activity was defined as themount of enzyme that released one mmol of p-nitroaniline per minnder the conditions of assay. Protein was determined by the methodf Lowry et al. (22) with bovine serum albumin (Bio-Rad) as therotein standard. The oxidation stability tests were examined withla-Ala-Pro-Leu-pNA (Peptide Institute) as substrate and H2O2 asxidant.SDS-PAGE was done on 12.5% acrylamide gels (Ready Gel-J;io-Rad) with 25 mM Tris-192 mM glycine buffer (pH 8.2) containing.1% SDS as the working buffer. Molecular masses were estimated

by SDS-PAGE with low-range molecular mass standards (Bio-Rad)atPAl

paAa

R

rbuIoaGcombpT6iPpatserAAwaetcE

ast1barto

us

aawnOGiw5bms

aAoio4nEpttqSpqsfabtspt

taTcrat

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

s molecular mass markers. Isoelectric points of enzymes were de-ermined on a Multiphore II gel electrofocusing system with a PAG-late and a Broad pI Calibration kit (Pharmacia) as pI markers.mino acid sequence alignments were done with a GENETYX Ma-

ign program (SDC Software Development).

Nucleotide accession numbers. The nucleotide sequences re-orted in this study have been submitted to the DDBJ, GenBank,nd EMBL data banks with Accession Numbers AB046402 for E-1,B046403 for KP-9860, AB046404 for LP-Ya, AB046405 for SD-521,nd AB046406 for NP-1.

ESULTS AND DISCUSSION

Purification and properties of the proteases. The pu-ified E-1 had a molecular mass of ;43 kDa, as judgedy SDS-PAGE, and had a specific activity of 115nits/mg protein at pH 10.5 in 50 mM borate buffer.soelectrofocusing analysis indicated that the pI valuef the enzyme was pH 9.7–9.9. The N-terminal aminocid sequence determined was Asn-Asp-Val-Ala-Arg-ly-Ile-Val-Lys-Ala-Asp-Val-Ala-Gln. In the absence of

alcium ions, caseinolytic activity of E-1 at 35°C wasbserved at a wide pH range from 6–12, with an opti-um pH around 10–11 in 20 mM glycine-NaOH

uffer. E-1 was stable at pH 6–12 but unstable belowH 5 and above pH 13 after a 24-h incubation at 25°C.he optimal temperature for activity at pH 10 was5°C. The enzyme was stable up to 65°C after a 30-minncubation at pH 10 in the presence of 5 mM CaCl2.henylmethylsulfonyl fluoride and diisopropyl fluoro-hosphate (1 mM each) inhibited the E-1 activity by 98nd 92%, respectively, a result that is consistent withhe classification of E-1 as a serine protease. Chelators,uch as o-phenanthroline, EDTA, and EGTA (5 mMach), did not inhibit the enzyme activity at all. Theelative ratio of activity toward Ala-Ala-Pro-Leu-pNA,la-Ala-Pro-Phe-pNA, Ala-Pro-Ala-pNA, and Ala-Ala-la-pNA was approximately 100:10:7:1. In contrastith E-1, typical subtilisins such as M-protease, H-221,nd BPN9 hydrolyzed Ala-Ala-Pro-Phe-pNA two- toightfold faster than Ala-Ala-Pro-Leu-pNA. Essen-ially, KP-9860, LP-Ya, SD-521, and NP-1 had physi-ochemical and enzymatic properties similar to those of-1 as described above.The striking feature of E-1, KP-9860, LP-Ya, NP-1,

nd SD-521, distinct from known subtilisins, is theirtrong resistance to oxidants such as H2O2, as well aso chelators such as EDTA and EGTA, as shown in Fig.. M-protease used as control was readily inactivatedy H2O2 under the same conditions. KP-9860, LP-Ya,nd NP-1, except for SD-521, were reported in theespective patents to be resistant to oxidative inactiva-ion. The results shown here are consistent with thexidative stability against sodium perborate of E-1.

Nucleotide and deduced amino acid sequences. Fig-re 2 shows the nucleotide and deduced amino acidequences of the representative E-1. The gene contains

315

single open reading frame (ORF), which begins withn ATG initiation codon at nucleotide 406 and endsith a TAA termination codon at 2311 in the 2478-bpucleotide sequence determined. Upstream of theRF, the putative ribosome binding sequence 59-AGGAG-39 is found, separated by 10 bp from the

nitiation codon. There is a promoter-like sequence,ith 59-TTTAGA-39 as the potential 235 region and9-TACATT-39 as the potential 210 region, separatedy 17 bp. A long inverted-repeat sequence (298.9 kJ/ol) from nucleotide 2323 to 2366 is found 9 bp down-

tream of the termination codon of the ORF.The ORF in the nucleotide sequence encodes 635

mino acids. The N-terminal amino acid sequence Asn-sp-Val-Ala-Arg-Gly-Ile-Val-Lys-Ala-Asp-Val-Ala-Glnf the native enzyme from strain D-6 was found in thenternal Asn1–Gln14. The calculated molecular massf the mature enzyme is, therefore, assumed to be5,636 Da, a value that is close to the 43 kDa of theative enzyme exoproduced by strain D-6. Thus, the-1 precursor has a possible 202-amino acid prepro-eptide. The prepro-enzyme has a potential signal pep-ide sequence, including a basic charged sequence athe N terminus, followed by a long hydrophobic se-uence with a possible signal peptidase cleavage site ofer(2175)-Gly(2174)-Ala(2173) (23). Between theredicted signal sequence and the mature enzyme se-uence, there is a very long hydrophilic sequence. Thisequence would be the pro-peptide, because the mostrequently conserved Val or Ile at 266, Val, Ile, or Phet 213, and Val, Ile, or Ala at 210 positions, countingackwards from the N termini of known mature sub-ilisins (24), are observed to be Val, Val, and Ile, re-pectively, at the same positions in the sequence. Thisredicted prepro-peptide sequence is approximatelywice as long as those of typical subtilisins.

FIG. 1. Rates of oxidative inactivation by H2O2 of the five pro-eases. Each enzyme was incubated in the presence of 50 mM H2O2

t 30°C and at pH 10 in 5.0 ml of 20 mM Britton-Robinson buffer.imed samples (0.5 ml) were withdrawn and mixed with 50 ml ofatalase solution (20 milliunits/ml) to remove residual H2O2. Theesidual activities of E-1 (F), KP-9860 (■), LP-Ya (h), SD-521 (Œ),nd NP-1 (‚), including M-protease (E), were measured by incuba-ion at 30°C for 10 min with Ala-Ala-Pro-Leu-pNA as substrate.

ious

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. Nucleotide and deduced amino acid sequences of the E-1 precursor. The deduced amino acid sequence of the gene product isndicated by the single-letter codes under the nucleotide sequence determined. The sequences similar to 235 and 210 consensus promotersf Bacillus are underlined. A putative ribosome-binding site is indicated by bold type. The ORF extends from Met(2202) to His433. Thenderlined amino acid sequence (Asn1–Gln14) refers to the N-terminal end of E-1 exoproduced by Bacillus sp. strain D-6. Convergent arrowshow an inverted repeat downstream of the stop codon TAA of the ORF.

316

4ehsFoh

aderimem

nawg

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Subtilisins are generally ;270 residues long. The33 residue-long mature E-1 includes a C-terminalxtension of ;160 residues (see Fig. 3). The extensionas a hydrophobic b-sheet structure and does not shareequence similarity with any residues reported to date.amily E kexins include endopeptidases with a varietyf C-terminal extensions (25), none of which shows anyomology with the C-terminal extension of E-1. We

FIG. 3. Multiple sequence alignment among the five oxidativelyumbered from the N-terminal residue of the mature enzyme and is inmino acids, Asp (electrophile), His (base), and Ser (nucleophile), is shith asterisks. Above the alignment, the sequences of structural eleiven. Underlines mark the positions of a-helices (H1–H10) and b-s

317

ttempted to truncate the C-terminal region by site-irected mutagenesis but failed to produce a mutantnzyme lacking only the C-terminal Pro424–His433esidue by E. coli cells (unpublished results). At leastn the case of E-1, therefore, the C-terminal extension

ay maintain the overall structural integrity of thenzyme, have long-range interaction with the secretionachinery of the host, or both.

able proteases and other subtilisins. Each amino acid sequence isated by the single-letter codes. A common catalytic triad of the threen by solid circles. Residues identical in all the sequences are markednts determined by X-ray crystallographic analysis of BPN9 (26) areds (e1–e9).

stdicowmetran

waaaLsoKhihtBse;miooaaLAtSvot(

Ep

TstsgaTqCsPiwaajwattbtcTSct

spBBBBNmp

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

To better understand the oxidant resistance of E-1,e sequenced the genes for KSM-9860, LP-Ya, SD-521,nd NP-1, deduced their amino acid sequences andligned them suitably, as shown in Fig. 3. The deducedmino acid sequence Asn-Asp-Val-Ala-Arg-Gly-Ile-Val-ys-Ala-Asp-Val-Ala is found commonly in the internalequences and coincides with the N-terminal sequencef mature E-1. The deduced amino acid sequences ofP-9860, LP-Ya, SD-521, and NP-1 showed very highomology to that of E-1 with 88.2, 98.6, 99.3, and 88.2%

dentity, respectively. However, the five enzymes ex-ibited very limited homology to those of true sub-ilisins, such as subtilisins Carlsberg with ;26% andPN9 with ;24% identity, high-alkaline proteases,uch as H-221 and M-protease with ;25% identity forach, and intracellular proteases with lower than23% identity. This low sequence homology is due toany insertions between the a-helices and b-strands

n BPN9, Carlsberg, H-221, and M-protease. The fivexidatively stable enzymes had practically no homol-gy with subtilisins in families B through F on themino acid sequence level. Nevertheless, when suit-bly aligned, the catalytic triads of E-1, KSM-9850,P-Ya, SD-521, and NP-1 are integrally conserved assp30, His71, and Ser254 (in E-1 numbering). Fur-

hermore, they have a Met residue next to the catalyticer residue. The presence of a Met residue in theicinity of the essential Ser is very puzzling becausexidative inactivation of subtilisins is caused by oxida-ion of this conserved Met to its sulfoxide derivative13, 14). We have already succeeded in crystallizing

FIG. 4. Unrooted phylogenetic tree of subtilisins inferred from thites. Conserved sequence segments around the catalytic triads (Asp3roteases were aligned manually with those of other subtilisins of knacillus sp. strain B189; D13158, Bacillus sp. strain AH-101; Q4552acillus alkalophilus PB92 (ATCC31408); Q99405, Bacillus sp. str21-2; P20724, Bacillus sp. strain YaB; Q45522, Bacillus sp. straacillus licheniformis; Q53521, B. licheniformis PWD-1 (ATCC 5375CIMB10278; P00783, B. subtilis var. amylosacchariticus; P04189,esentericus; P00781, Bacillus amyloliquefaciens; P11018, B. s

olymyxa 72.

318

-1 and are now solving its tertiary structure to ex-lain the mechanism of the oxidative stability.

A phylogenetic tree and evolutionary relationship.he deduced amino acid sequences of the five proteaseshown in this study were aligned and compared withhose of 24 subtilisins categorized in family A. Allequences were incorrectly aligned, whereas the re-ions around their catalytic residues (Asp32, His64,nd Ser221 in BPN9 numbering) are well conserved.his is due to low similarity of the gene products se-uenced in this study to other subtilisins and to the-terminal long amino acid extension. Thus, consensusequence segments of the 29 proteases, Tyr21–Lys42,ro57–Ala92, and Asn212–Leu235 (in BPN9 number-

ng), were manually aligned, and the aligned segmentsere used to construct a phylogenetic tree. Evolution-ry distances among the proteases were computed, andphylogenetic tree was constructed by a neighbor-

oining algorithm, as shown in Fig. 4. The 29 proteasesere unequivocally grouped into four clusters based onmino acid sequence analysis. Two major clusters arehe groups of true subtilisins and highly alkaline pro-eases. Each cluster was composed of 10 or 11 mem-ers, and some subclasses were constituted in the clus-ers. Three enzymes, AML1, ispA, and INT72, werelustered and categorized as intracellular proteases.he fourth cluster is composed of E-1, KP-9860, LP-Ya,D-521, and NP-1, and two subclasses were furtheronstituted in this cluster. This cluster is quite dis-antly related to the other three clusters, suggesting

ino acid sequence alignment of the conserved regions around activeHis64, and Ser221 in BPN9 numbering) of the five oxidatively stable

sequence. Bar is Knuc unit. Sources of sequences aligned: Q45521,acillus sp. strain NKS-21; P41362, Bacillus sp. strain 221; P27693,KSM-K16; P29599, Bacillus lentus; AB005792, Bacillus sp. strain-825-6; Q45466 and Q45467, Bacillus sp. strain LG12; P007780,P00781, Bacillus subtilis DY; P29142, Bacillus stearothermophilussubtilis 1168; P35835, B. subtilis (natto) NC 2-1; P07518, Bacillusilis IFO3013; Q998944, B. amyloliquefaciens; P29139, Bacillus

e am2,

own3, Bainin G7);B.

ubt

that the enzymes in the cluster may have some specialpbs

tnspe

R

1

1

12. Masui, A., Fujiwara, N., and Imanaka, T. (1994) Stabilization

1

1

1

1

1

1

1

2

2

2

2

2

2

2

Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

roperties. Actually, these five enzymes are highly sta-le to oxidants as one of common properties, as de-cribed above.In conclusion, we propose that E-1 and other oxida-

ively stable, alkaline proteases be categorized as aew class of subtilisins, distinctly different from trueubtilisins, highly alkaline proteases (high-alkalineroteases), intracellular proteases, and other relatednzymes.

EFERENCES

1. Rao, M. B., Tanksale, A. M., Chatge, M. S., and Deshpande, V. V.(1998) Molecular and biotechnological aspects of microbial pro-teases. Microbiol. Mol. Biol. Rev. 62, 597–635.

2. Siezen, R. J., and Leunissen, J. A. M. (1997) Subtilases: Thesuperfamily of subtilisin-like serine proteases. Protein Sci. 6,501–523.

3. Horikoshi, K. (1971) Production of alkaline enzymes by alkalo-philic microorganisms. Part I. Alkaline protease produced byBacillus no. 221. Agric. Biol. Chem. 35, 1407–1414.

4. Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S.,and Wilson, K. S. (1992) Crystal structure of the alkaline pro-teinase Savinase™ from Bacillus lentus at 1.4 Å resolution. J.Mol. Biol. 223, 427–445.

5. Zuidweg, M. H. J., Bos, C. J. K., and van Welzen, H. (1972)Proteolytic compounds of alkaline proteases of Bacillus strains.Zymograms and electrophoretic isolation. Biotechnol. Bioeng. 16,685–714.

6. Kobayashi, T., Hakamada, Y., Adachi, S., Hitomi, J., Yoshi-matsu, T., Koike, K., Kawai, S., and Ito, S. (1995) Purificationand properties of an alkaline protease from alkalophilic Bacillussp. KSM-K16. Appl. Microbiol. Biotechnol. 43, 473–481.

7. Van der Laan, J. M., Teplyakov, A. V., Kelder, H., Kalk, K. H.,Misset, O., Mulleners, L. J. S. M., and Dijkstra, B. W. (1992)Crystal structure of the high-alkaline serine protease PB92 fromBacillus alcalophilus. Protein Eng. 5, 405–411.

8. Yamane, T., Kani, T., Hatanaka, T., Suzuki, A., Ashida, T.,Kobayashi, T., Ito, S., and Yamashita, O. (1995) Structure of anew alkaline serine protease (M-protease) from Bacillus sp.KSM-K16. Acta Cryst. D51, 199–206.

9. Shirai, T., Suzuki, A., Yamane, T., Ashida, T., Kobayashi, T.,Hitomi, J., and Ito, S. (1997) High-resolution crystal structure ofM-protease: Phylogeny aided analysis of the high-alkaline adap-tation mechanism. Protein Eng. 10, 627–634.

0. Wells, J. A., and Estell, D. A. (1988) Subtilisin—An enzymedesigned to be engineered. Trends Biochem. Sci. 13, 291–297.

1. Siezen, R. J., de Vos, W. M., Leunissen, J. A. M., and Dijkstra,B. W. (1991) Homology modelling and protein engineering strat-egy of subtilases, the family of subtilisin-like serine proteases.Protein Eng. 4, 719–737.

319

and rational design of serine protease AprM under highly alka-line and high-temperature conditions. Appl. Environ. Microbiol.60, 3579–3584.

3. Stauffer, C. E., and Etson, D. (1969) The effect on subtilisinactivity of oxidizing a methionine residue. J. Biol. Chem. 244,5333–5338.

4. Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B., andPower, S. (1988) The three-dimensional structure of Bacillusamyloliquefaciens subtilisin at 1.8 Å and an analysis of thestructural consequences of peroxide inactivation. J. Biol. Chem.263, 7895–7906.

5. Estell, D. D., Graycar, T. P., and Wells, J. A. (1985) Engineeringan enzyme by site-directed mutagenesis to be resistant to chem-ical oxidation. J. Biol. Chem. 260, 6518–6521.

6. Hakamada, Y., Kobayashi, T., Hitomi, J., Kawai, S., and Ito, S.(1994) Molecular cloning and nucleotide sequence of the gene foran alkaline protease from the alkalophilic Bacillus sp. KSM-K16. J. Ferment. Bioeng. 78, 105–108.

7. Saito, H., and Miura, K. (1963) Preparation of transformingdeoxyribonucleic acid by phenol treatment. Biochim. Biophys.Acta 72, 619–629.

8. Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Plainview, New York.

9. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang,Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST andPSI-BLAST: A new generation of protein database search pro-grams. Nucleic Acids Res. 25, 3389–3402.

0. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) ClustalW: Improving the sensitivity of progressive multiple sequencealignments through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.

1. Saitou, N., and Nei, M. (1987) A neighbor-joining method: A newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol. 44,406–425.

2. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, J. R.(1951) Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193, 265–275.

3. Simonen, M., and Palva, I. (1993) Protein secretion in Bacillusspecies. Microbiol. Rev. 57, 109–137.

4. Siezen, R. J., Leunissen, J. A. M., and Shinde, U. (1995) Homol-ogy analysis of the propeptides of subtilisin-like serine proteases(subtilases). In Intramolecular Chaperones and Folding (Shinde,U., and Inouye, M., R. G. Eds.), Chap. 11, pp. 233–256, LandesCompany, Austin.

5. Seidah, N. G., and Chretien, M. (1994) Pro-protein convertases ofsubtilisin/kexin family. Methods Enzymol. 244, 152–167.

6. Gallagher, T., Gilliland, G., and Bryan, P. (1995) Theprosegment-subtilisin BPN9 complex: Crystal structure of a spe-cific ‘foldase’. Structure 3, 907–914.