JOURNAL OF BACTERIOLOGY, May 1991, p. 3078-3083 0021-9193/91/103078-06$02.00/0 Copyright C 1991, American Society for Microbiology

Vol. 173, No. 10

Xylose (Glucose) Isomerase Gene from the Thermophile Thermus thermophilus: Cloning, Sequencing, and Comparison with Other Thermostable Xylose Isomerases KOEN DEKKER,"2 HIDEO YAMAGATA,l KENJI SAKAGUCHI,2 AND SHIGEZO UDAKA1* Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464,1 and Nihon Shokuhin Kako Co. Ltd., Marunouchi, Chiyoda-ku, Tokyo 100,2 Japan Received 27 November 1990/Accepted 5 March 1991

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces gniseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophUus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermnophilus. Comparison of the Thermnus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermnus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.

D-Xylose isomerase (EC 5.3.1.5) catalyzes the reversible isomerization of D-xylose to D-xylulose in the first step of xylose metabolism following the pentose phosphate cycle. It also catalyzes the isomerization of glucose to fructose. Therefore, it is used industrially in the production of highfructose corn syrup under the name glucose isomerase (13). The isomerization is reversible, and the final fructose content depends on the reaction temperature. Use of a higher temperature results in a higher fructose content. The enzyme has been isolated from many microorganisms and has been well studied (6, 7). Genes from Escherichia coli (29), Bacillus subtilis (35), Clostridium species (10, 17), Streptomyces species (11, 15), Ampullariella species (27), and Actinoplanes missouriensis (2) have been sequenced. The crystal structure of Actinomycetes xylose isomerase, an (a/p)8 barrel, has been resolved (5, 8, 26), and the reaction mechanism has been gradually unraveled (9, 17). Since thermophiles such as Thermus species are known to produce highly thermostable enzymes (24), it is interesting to analyze the structure and thermostability of xylose isomerases from thermophiles. Lehmacher and Bisswanger (18, 19) recently purified xylose isomerase from Thermus species and showed that the xylose isomerase is indeed exceptionally stable at elevated temperatures. After 30 days at 70°C, glutaraldehyde-cross-linked Thermus xylose isomerase retained 100% activity (18), while similarly treated Streptomyces xylose isomerase retained about 25% activity (14). However, the amount of enzyme produced by Thermus species is too small. This article describes the cloning and analysis of the xylose isomerase gene and the efficient production of the enzyme in E. coli. The amino acid sequence of this ex*

tremely stable xylose isomerase was compared with sequences of other thermostable xylose isomerases to see if any amino acid substitutions could be found which might affect the irreversible inactivation at high (>80°C) temperatures. MATERIALS AND METHODS

Strains and plasmids. E. coli JM109 was grown in 2YT (33) at 37°C and transformed by the method of Hanahan (12). Thermus thermophilus HB8 (kindly supplied by T. Oshima) (25) was grown at 70°C by the method of Tanaka et al. (32) with xylose as a carbon source. T. thermophilus HB8 (ATCC 27634) is listed in recent ATCC catalogs as Thermus aquaticus; however, numerical classification by Santos et al. (28) places T. aquaticus and T. thermophilus in two different clusters. Therefore, the older name, T. thermophilus, is used here. The Streptomyces griseofuscus xylose isomerase gene was kindly provided by C. Fukazawa (15). DNA preparation and sequencing. Genomic T. thermophilus DNA was extracted as follows. A frozen cell pellet (1 g) was suspended in 10 ml of 50 mM Tris-HCl (pH 8)-200 mM NaCl-100 mM EDTA and lysed with 1% sodium dodecyl sulfate (SDS) (15 min, 60°C). The lysate was treated with RNase A (0.05 mg/ml, 30 min, 60°C) and proteinase K (0.05 mg/ml, 60 min, 60°C). Debris was removed by centrifugation, and the clear lysate was extracted four times with phenol. After the addition of 0.8 volume of isopropanol, DNA was spooled out, dissolved in 50 mM Tris-HCl (pH 8)-150 mM NaCl-1 mM EDTA, and purified on a DEAE column. For sequencing, overlapping fragments were subcloned in pUC118 or pUC119. Single-stranded DNA was prepared by the method of Vieira and Messing (33) and sequenced with Taq DNA polymerase by using 7-deazadGTP.

Corresponding author. 3078

VOL. 173, 1991

XYLOSE ISOMERASE GENE FROM T. THERMOPHILUS

Protein sequencing. Partially purified xylose isomerase from E. coli was separated by SDS-polyacrylamide gel electrophoresis, eluted, and sequenced on a Applied Biosystems 477A protein sequencer. Comparison of amino acid sequences. Amino acid substitutions in aligned sequences of Streptomyces violaceoniger (11), A. missouriensis (2), Ampullariella species (27), and T. thermophilus were scored by the method of Argos et al. (3, 21). Details concerning the calculation were described by Argos et al. (3). First the amino acid changes (a., where a, is the number of substitutions of amino acid i in all three Actinomycetes sequences by amino acidj in T. thermophilus were scored. From this, the net traffic between amino acid i in Actinomycetes species to amino acid j in T. thermophilus was calculated. For example, by using Table 2, one can see that the net traffic of glycine-to-proline substitutions is five (in Argos et al. [3], D = a- aji 5 - 0 =- 5). The sum of all such substitutions (lia. - ilaji = ilDW) gives the preference of T. thermophilus for a certain amino acid. For example, the preference of T. thermophilus for proline follows from all substitutions by proline (i7aip = 28) minus all substitutions of proline (ilap, = 21). A negative ila, j, means that a particular amino acid j is frequently replaced by another amino acid in T. thermophilus. Enzyme assay. Enzyme activity was measured by incubation of 0.1 to 0.4 U of xylose isomerase in 1 ml of 100 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7)-400 mM fructose-10 mM MnCl2 at 85°C. Glucose production was followed by analysis of 20-1.I1 samples with a glucose oxidase-based assay (glucose B-test; Wako). One unit was defined as the amount of enzyme that produces 1 ,umol of glucose per min under these conditions. Enzyme isolation. E. coli cells (0.5-liter culture) were induced at mid-log phase by the addition of 1 mM isopropylP-D-thiogalactopyranoside, grown for more 2.5 h, and harvested by centrifugation. Thermus cells (5-liter culture) were harvested in late logarithmic phase. Washed cells were resuspended in 100 mM HEPES (pH 7)-10 mM MnCl2-0.05 mM phenylmethylsulfonyl fluoride (a protease inhibitor) and disrupted by sonication or by use of a French press, and the debris was spun down. The supernatant was heated for 10 min at 85°C in the presence of 10 mM Mn2' and allowed to cool. Denatured proteins were removed by centrifugation (18,000 x g, 10 min, 4°C). The supernatant was dialyzed two times against 500 volumes of 50 mM HEPES (pH 7)-5 mM EDTA and two times against 500 volumes of the buffer without EDTA. Nucleotide sequence accession number. The DDBJ, EMBL, and GenBank accession number for the sequence reported in this article is D90256. RESULTS Cloning and sequencing. Genomic DNA from T. thermophilus was digested with SacI and fractionated on a 0.4% agarose gel and electrotransferred to a nylon membrane. Southern hybridization at 54°C with a 287-nucleotide BglI fragment from the S. griseofuscus xylose isomerase gene (15), corresponding to nucleotides 700 to 987 in T. thermophilus (Fig. 1), gave a single positive band of about 1.8 kb. Thus, only one copy of the gene was present in the T. thermophilus genome. Genomic Sacl fragments of about 1.8 kb were isolated from agarose and ligated with SacI-digested pUC118. Colony hybridization of transformed E. coli identified several positive colonies. Southern hybridization on SacI-digested plasmid DNA from these colonies showed that

3079

they contained a 1.8-kb fragment hybridizing with the S. griseofuscus probe. This fragment was sequenced in both directions, showing an open reading frame coding for the putative T. thermophilus xylose isomerase (Fig. 1). The translation start (GTG) was confirmed by protein sequencing of the NH2 terminus. The possible ribosome binding site is located 8 nucleotides before the translation start; further upstream is an AT-rich region. As in other Thermus genes, consensus -10 and -35 regions are not obvious (16, 22, 23). A possible transcription terminator (AG = -130 kJ/mol or -31 kcallmol) is found 150 nucleotides downstream of the translation stop codon. Comparison of several xylose isomerase amino acid sequences. Homology between the T. thermophilus coding sequence and other published sequences is summarized in Table 1. Comparison of amino acid sequences (Table 1) is more appropriate than comparison of nucleotide sequences because of the difference in G+C content of the compared strains, e.g., 66% for T. thermophilus, 36% for B. subtilis, 49% for E. coli, and 71% for S. violaceoniger. The amino acid sequences were aligned to see if any homologous regions exist and to determine what kind of amino acid substitutions accompany increased thermostability. When all sequences were compared, relatively few regions of homology were found (Fig. 2). However, the amino acids involved in substrate binding and isomerization (5, 8, 9) and the spacing between them are well conserved. These residues are not situated in one domain, but they are spread out along the primary structure. Apparently, in spite of the low homology, the sequences form similar tertiary structures. When the amino acid sequence of Thermus xylose isomerase is compared with the sequences of the thermostable Actinomycetes xylose isomerases (2, 11, 15, 27), homology is high. This comparison was done to identify amino acid substitutions characteristic for the more stable Thermus isomerase. All substitutions are summarized in Table 2 by using the method of Argos (3, 21; see Materials and Methods). Some amino acids are preferred by T. thermophilus, while some amino acids are less frequently found in T. thermophilus than in Actinomycetes species. As a result, one can rank the amino acids by preference in the more thermostable Thermus xylose isomerase (the value of ila - ilajl is shown between branches). Ranking the preferred amino acids (positive ilay - iSaj1) results in the following: E(30) > R(22) > Y(19) > V(18) > P, M(7) > L(4), C(2), N, K, W(1). The relatively sparse amino acids (negative i7Eaij - ilaj,) resulted in the following ranking: D(-27) > I(-21) > G, T(-17) > Q(-11) > A(-9) > H, S(-4), F(-2). If one considers only substitutions that involve amino acids which are conserved between the other species, but which are different in T. thermophilus, the ranking is similar except that isoleucine appears to be more preferred and leucine and arginine appear to be less preferred. The net traffic (calculated from Table 2, Dii = a1 - aj,; see Materials and Methods) indicates which less preferred amino acid is replaced by which preferred amino acid. The most frequent substitutions of the least preferred (i7Xa, - lEa1. < -10) and by the most preferred (lEav - lZafl> 10) amino acids are schematically shown in Fig. 3. Expression of the Thermus xylose isomerase gene. The Thermus xylose isomerase gene was cloned into plasmid pUS12 (30) for expression in E. coli under control of the strong, inducible tac promoter as follows. The Thermus xylose isomerase gene from the BbeI site (position 231 in the sequence, 57 nucleotides upstream from the translation start

3080

J. BACTERIOL.

DEKKER ET AL. 10

20

30

40

T0

*0

90

100

200

60

910

120

210

930

940

950

TF 0C L N P F F A H 8 TC 990 1000 1010 980

960 A

G

L

1020

TTAACTTT0YSCAcGCCGT00CCCAOGCTCTcGAC0cc000AAGCTTTTCCACATTGACC N F V H AV A Q A L D A G K L F H I D L

270 290 300 260 280 00p u.pp p ua MT0?OTM0?ACGTMXWMA Y AR-rh58 P a 350 310 320 330 340 360 P a T FV aLVYT V O N V a R D P F 310 380 400 390 410 420 -~GARE 0TC0G0AGC004cO0TTc0OGTATAA0CTG0G0A0GC a D A V R I B L D P V Y V V H K L A F L

260

1030 1040 1050 1060 1070 1080 TCAACGACCACGGATGAGCCOOTTGACCAGGACCC GCTCGAGAACCTCA N D Q R M S R F D Q D L R F G 8 e N L K

1110 1090 1100 1120 1130 1140 AGOCOGCCTTT?TCCTOOTOGACCTCCTOGAAAGCTCCGGCTACCAOGGCCCCCGCCACT A A F F L V D L L E SS G Y Q G P R H F 1150

1160

1170

1180

1190

1200

TTGACGCCCACGCCCTGCGTACCGAGGACAGAAGGOGTTTGGGCCTTCGCCCGAGGCT D A U A L R T E D F E G V W A F A R G C

430

440

450

460

470

480

1210 1220 1230 1240 1250 1260 GCATOCOTACCTACCTGATCTTAAAGGAAAOGGCTGAAGCCTTCCGCGAGGATCCCGAGG M R T Y L I L K E R A E A F R E D P E V

490

500

510

520

530

540

1300 1270 1280 1290 1310 1320 TCAAGGAGCTTCTTGCCGCTTACTATCAAGAAGATCCTGCGGCCTTGGCCCTTTTGGGCC K E L L A A Y Y Q e D P A A L A L L G P

TTGOOCCAcOGOTAAcTICGOAcAGOCCTATcCGCOOGACGCCTCCTC a A T a v N L N D 9 D L I P R a T P P Q AT A A~0C AAGG AGAGCMGACC O CTT I R D Q I V 2 R F K A L D 8 T G L K V 550 560 590 600 570 S80 TCGCCAV_OTCAc_OcCAA_CTCTT_TccOAccCTOCTTTCAAGGACGOGGCCTACGA P N V T- A N

610

L

620

P D P V V 2

670 I

B

970

CWAA0DCq

A

920

D R P

180 1T0 16" ------CTOAGOTOCATOAOCCC 220 230 240

10 130 140 TC?AOAOOGOTOCGG AOOCGOGCTTTCCCO

190

50 110

L a

730

F

8

D

F

K

D G

P

T

650

640

630

A

A

T A

I

E

L

B

K 8

690

740

T

V

V

I

L

T

660

V

P G B

750

M D

L

710

700

a A

9

760

R

v

770

T a K A 2 K V W D W V R

A

T A 8

790 850

oO, D Q 860

810

a T a T 2

820 F

870

A

890

880

N

1360

1370

1380

1390

1400

1410

1420

1430

1440

G

1460 1500 14S0 1470 1480 1490 TGCGOG0GTGAGGCGGCCATCGGCTTOACCTGGGAACGAGCGGGCTCAAGGCCCTGGT

R G

8

1S20 IS80

1S40

1ss0

1590 1600 CGAGOCCGGOCTGGACGGAGCAGGACCCCCAGGAC 1650 1640. 1630 1660

1610 16 0

1510

1530

1560 1620 AGGTGT 1680 -----TO AGGTGGTGOGCCTGGGGCTTTCCGGGC Tccgggaaacsacco

GCTGGACGAGGAGGGOTAGAAAGCGCGCTGAGGCCCGGCCGGTTACCCCCTTCACACCC

IS70

840 P

1350

720 780

830

8 P X

L

1340

GGCGCCGCGGTTATGCCCTGGAACGCCTGGACCAGCTGGCOGTGGAGTACCTCCTGGGGG R R G Y A L E R L D Q L A V E Y L LG V

TTVATLA E A L N F N A

A

1330

CCTACTCCCGCGAGAAGGCCGAAGCCCTCAAGCGGCGCGAGCTTCCCCTCGAGGCCAAGC Y 8 R E K A E A L K R A E L P L E A K R

8

ATOCC?TOcOOAAGAOcCTOOAOACCATGGACCTG

680 A

P A

1700 1710 1730 1740 1s9 1720 AGATGCACOGOOCGGTCTTCCTGGACCGGGAGGGCCGTTTCCTCCTTCCTGCGCCCCTTT 1760 1750 1770 1800 1780 1790 OGAACGACCAGCOCACGGAGGAGGAAGTCCOGTGGATGGAAGAGGTCTTCCCTCGGCCCG

8 900

a_0O0 G ACCA TOGGAGCATGCTC0CCTTTATTCATACCC P R a D I Y F A T V a tN L A F I H T L

FIG. 1. Nucleotide and deduced amino acid sequence of the T. thermophilus glucose isomerase gene. The possible ribosome binding site (RBS) and an AT-rich region upstream of the ribosome binding site are underlined. The NH2-terminal amino acids that were confirmed by protein sequencing are also underlined. A possible transcription terminator is indicated by inverted arrows.

site at 288) to the SmaI site (position 1534 in the sequence, 83 nucleotides downstream from the translation stop site at 1451) was inserted between the BamHI and SmaI sites of pUS12, resulting in expression vector pUSTXI (BamHII BbeI connection in pUSTXI is GGATCCCCCTTGG). Thus,

expression of the xylose isomerase gene on pUSTXI is under control of the tac promoter but uses the Thermus ribosome binding site and translation start site. After transformation of E. coli JM109 with pUSTXI, xylose isomerase levels reached 450 U/liter during the sta-

TABLE 1. Relative homology between nucleotide and amino acid sequences from several microorganisms amino

nucleotides

acids

M()

T.t

S.g

S.v

A.m

56

58

55

54

29

27

27

26

96

66

65

27

27

27

24

68

67

27

27

27

25

93

26

30

27

27

27

27

27

26

86

70

50

71

52

A.

C.ts C.th B.s

E.c

(X) T. thermophi1us

S.griseofuscus

65

S. violaceoniger

65

98

A .missouriensis

63

73

75

Ampullari ella

62

74

75

92

C. thermosul furogenes

44

46

47

45

44

C. thermohydrosul furicuis

43

43

42

44

44

78

B. subtilis

43

45

44

44

47

68

69

E. col i

45

47

47

46

45

56

57

51 59

XYLOSE ISOMERASE GENE FROM T. THERMOPHILUS

VOL. 173, 1991

1 1 1

1

I T.t I 2 S. I 3 S.v I

4 A.m 5 A

I

1

6 C.tal 7 C.thl

1

8

I

9 E.c

1

MYEPKPYER NSFQPTPHDK MSFQPTP8DK NSVQATREDK MSLQATPDDK MNKYFENVSKIKYEFGPSNNPYSFKFYNPEEVIDGKTEEHLR NEYFKNVPQIKYEGPKSNNPYAFKFYNPDE IIDGKPLKEHLR

B.8 l

MAQSHSSSVNYFGSVNKVVFEGKASTNPLAFKYYNPQEVIGGKTHKKNLR MQAYFDQLDRVRYEGSKSSNPLAFRHYNPDELVLGKRMEEHLR 25

25

0

FTFGLV-TVGNVGRDL

1

1

81 FSIAYWHTFTADGTD

1 1

1 1

I

TMQPWDHY-KGMDLAUARVEAAFENFEKL---DAPFFAF

91 FAACYWHTFCVNGA VGAFNRPWQQPGEALALAKRKADVAFEFFHIKL---HVPFYCF 53 56 89,90 93 DPAFKDGAFTSPDP I 1l lIPRGTPPQERDQIVRRFKKAL----DETGLK 1 PVFKD-RFTANDR 2 NPFGSSDTERESHIKRFRQAL----DATGMTV PVFEDGGFTANDR 1 IPFGSSDTERESHIKRFRQAL ----DATGTVP 131 HPVFKDGGFTSNDR 4FGSDAQTRDGI IAGFKKAL----DETGLIVP I4 l DIVPFCADAATRWGIVAGFSKAL----DETGLIV I HPVFKDGGFTSNDR

16ln APEGETLRETNKNLDTIVANIKDYLKTSKTKVLV APEGDTLRETNKNLDTIVAMIKIYLKTSKTKVLV

1 1

1 1

1 1 1 1

1 l 8 b APEGSTLKETNQNLDI IVGFIIKDYMRDSNVKLLV I91} bSPEGASLKEYINNFAQMVDVLAGKQEESGVKLLW

6)

KEIGFEGQFLI

71 QEIGFEGQFLI 81 KEIEYTGQFLT 91 HKIGFQGTLLI

NEPRGDIYFATVGSMLAFIHTLDRPERFGLNPEFAHEThAGLNFV NEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQt4AGLNFP

EPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFP

NEPRGDILLPTAGHAIAFVQELERPELFGINPETGHEQMSNLNFT

NEPRGDILLPTAGHAAIAFVQELERPELFGINPETGHEQNSNLNFT IEPTKHQYDFDVA?"'LAFLRKYDLDKYFKVNIEANHATLAFHDFQ EPTKHQYDFDAASVHAFLKKYDLDKYFKLNIEANHATLAGHDFQ EPTTHQYDTDAATTIAFLKQYGLDNHFKLNLEANHATLAGHTFE EPTKHQYDYDAATVYGFLKQFGLEKEIKLNIEANHATLAGHSFH 254,256

244 111

HAVAQALDAGKLFHI

21 HG I AQALWAGKLFHI

1

31 HGIAQALWAGKLFHI

1

41 151

1

61

1

71

1

81

91 11

FGSENLKAAFFLVDLLE----SSGY-QG

:=N-US(II

RFAAGDLRAAFWLVDLLE ---- SAGY-EG FGAGDLRAAFWLVDLLE-----SAGY-EG N-GQSGIKY VFGHGDLLNAFSLVDLLENGPDGAPAY-DG QGIAQALWHKKLFH 'N-GQHGPKF QGIADALWHKKLFHI4N-GQHGPK VFGHGDLLNAFSLVDLLENGPDGGPAY-DG HELRYARINGVLGS NTGDNLLG QFPTDIRMTTLAMYEVIK -----MGGFDKG QYPTDIRMTTLAMYEVIK-----MGGFNKG HELRYARINNMLGSI NNDMLLG HELRMARVHGLLGS EFPTDLYSTTLAMYEI LQ-----NGGLGSG NQGHPLLG FPNSVEENALVMYEILK ---- AGGFTTG HEIATAIALGLFGS NRGDAQLG 286 PRH FlIALR--TEDEEGVWAFARGCMRTYLILKERAEAFREDPEVKEL-LAAYYQEDPA PRH FKPPR--TEDFDGVWASAEGCMRNYLILKQPAAAFRADPEVQEARVGAARLDQLA

1

NPRFVHGASTSCNA

1

NPRFVHGAATSCNA

1

21 31 PRH FKPPR--TEDFDGVWASAEGCKRNYLI LKERAAAFRADPEVQEA-LRAARLDQLA 41 PRH FMflPSR--TEDYDGVWESAKANIRMYLLLKERAKAFRADPEVQEA-LAASKVAEIK 51 PRH lKPSR--TEDFDGVWESAKDNIRMYLLLKERAKAFRADPEVQAA-LAESKVDELR 61 GLN KVRRASFEPED-LFLGHIAGNDAFAKGFKVAYKLVKDRVFDKF-I-EERYASYK GLN lVRASFEPED-LFLGHIAGMDAFAKGFKVAYKLVKDGVFDRF- I-EERYKSYR 81 GLN KVRRSSFEPDD-LVYAHIAGMDAFARGLKVAHKLIEDRVFEDV-I-QHRYRSFT

1

91 GLN

1 1 1

1

NPRFVHGAATSCNA NPRYGAGAATNPDP

7

11 EDQGYGYRFA 21 TAQGYDLRFAI 31 TAQGYDLRFA! 4) EDRGYGLRFAI 51 EDQGYGLPFAI

0

RDAV--RERLDP--------------VYVVHKLAELGAYGVNL 1l 21 FTFGLW-TVGWQGRDI WDAT--RPGLDP--------------VETVQRLAELGAYGVTF 31 FTFGLW-TVGWQGRDI WDAT--RPALDP--------------VETVQRLAELGAYGVTF 4l FSFGLW-TVWQARD DAT--RTALDP--------------VEAVHKLAEIGAYGITF 5l FSFGLW-TVGWWDt 3DAT--RPVLDP---------I-----IAVHKLAEIGAYGVTF 61 FSIAYWUTFTATw SKAPWNHYTDPNDIAKARVEAAFEFFDKI---NAPYFCF 71 FSVAYVTFTANGTD PThQUPVDHFTDPNDIAKARVEAAFELFEKL---DVPFFCF

1

1

3081

1

VRRQSTDKYD-LFYGHIGAMDTNALALKIAARMIEDGELDKR-I-AQRYSGWN

136 .~~~~~~~16 .. ..

1 1

1 1

1 1 1

I

..

11 WRAYALRKSLETMDLGAELGAEI WVGREGAEVEATGKARKVWDWVREALNFMAAYA 21 DVRAYAVRKTIRNIDLAAELGAKTYV GREGAESGGAKDVRDALDRMKEAFDLLGEYV REGAESGGAKDVRDALDRMKEAFDLLGEYV 31 DVRRYALRKTIRNIDLAAELGAKTYVW 41 SVRRYAIRKVLRQNDLGAELGAKTLV qGREGAEYDSAKDVSAALDRYREALNLLAQYS 51 SVRRYAIRKVLRQNDLGAELGAKTLV ;WREGAEYDSAKDVGAALDRYREALNLLAQYS REGYETLLNTDMEFELDNFARFLHMAVDYA 61 DVFAYSAAQVKKALEITKELGGENYV 71 DVFAYAAAQVKKALEITKEGITENYGN REGYETLLNTDMEELDNFARFLHNAVEYA REGYETLLNTDLKFELDNLARFMHNAVDYA 81 DVFAYAAAQVKKGLETAKELGAENYV GREGYETLLNTDLRQEREQLGRFMQNVVEHK 91 EVFSWAATQVVTAMEATHKLGGENYV

1

1 1

1

11 21 31 41

1

51 61 71 81

1

91

1 1 1

ALA--LLGPY--SREKAEALKRAELPLE-AKRRRGYALERLDQLAVEYLLGVRG QPT--AADGL--EALLADRTAFEDFDVEAAAARAAW-FERLDQLAMDHLLGARA QPT--AADGL--EALLADRTAFEDFDVEAAAARAAWPFERLDQLAMDHLLGARG TPTLNPGEGY--AELLADRSAFEDYDADAVGAK-GFGFVKLNQLAIEHLLGAR TPTLNPGETY--ADLLADRSAFEDYDADAVGAK-GYGFVKLNQLAJDHLLGAR DGI--GADIVSGKADFRSLEKYALERS-QIVNKSGRQ-ELLESILNQYLFAE EGI--GAEIVSGKANFKTLEEYALNNP-KIENKSGKQ-ELLESILNQYLFSE EGI--GLEITEGRANFHTLEQYALNNK-TIKNESGRQ-ERLKPILNQ SEL--GQQILKGQMSLADLAKYAQEHHLSPVHQSGRQ-EQLENLVNHYLFDK

FIG. 2. Comparison of the xylose isomerase amino acid sequences of T. thermophilus [1], S. griseofuscus (corrected) [2], S. violaceoniger [3], A. missouriensis [4], Ampullariella species [5], Clostridium thermosulfurogenes [6], Clostridium thermohydrosulfuricum [7], B. subtilis [8], and E. coli [9]. Numbers are for the T. thermophilus enzyme. Boxes indicate active-site amino acid residues. Dots mark amino acids which are conserved in all nine sequences compared. Open circles mark amino acids that are conserved in sequences 2 to 9 but are different in T. thermophilus.

similar. This suggests that no mutations have occurred during cloning of the gene. Purification of the enzyme produced by E. coli was rather straightforward. Heat treatment at 85°C for 10 min of cell extract gave a 20-fold purification. The Thermus xylose

tionary phase, roughly a 45-fold increase compared with production by T. thermophilus (Table 3). Endogenous activity of E. coli cell extract after heat treatment was negligible. The characteristics, e.g., temperature optimum, of the enzyme produced in E. coli and in T. thermophilus are

TABLE 2. Amino acid substitutions when S. violaceoniger, A. missouriensis, and Ampullariella xylose isomerases T. thermophilus xylose isomerase (see Materials and Methods) R

N

D

70 9 8 51

2

1

A A R a

C

D C

i

e

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3082

J. BACTERIOL.

DEKKER ET AL.

Thermus

actinomycetes v

-

K

IR

I

V

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A-

FIG. 3. Schematic representation of the most frequent amino acid substitutions between Actinomycetes and Thermus xylose isomerase.

isomerase is stable under these conditions. After heat treatment, the enzyme was about 60% pure as judged by comparison with the specific activity of enzyme purified by Lehmacher and Bisswanger from Thermus species (18). Estimation of the subunit molecular weight by SDS-polyacrylamide gel electrophoresis indicated a molecular weight of 44,200, which is in reasonable agreement with the calculated molecular weight of 43,900. Gel filtration on Superose 12 (Pharmacia, Uppsala, Sweden) showed a molecular weight of about 200,000. This suggests that Thermus xylose isomerase, like most known xylose isomerases, is a homomeric tetramer. DISCUSSION T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and sequenced. The Thermus xylose isomerase is more thermostable than any other known xylose isomerases (14, 18) and may therefore be useful in the production of high-fructose corn syrups with higher fructose contents (13). Thermus xylose isomerase is also interesting for the study of thermostability and thermoinactivation. Thermoinactivation is generally described as a two-step process: (i) reversible unfolding, followed by (ii) irreversible inactivating reactions (native

=

unfolded

-*

inactive) (1).

Is the increased thermostability of Thermus xylose isomdue to an increase in stability against reversible unfolding (step i) or is it due to replacement of amino acids which are prone to react during thermoinactivation (step ii)? Argos et al. (3, 21) compared enzymes from mesophiles with enzymes from thermophiles and showed that, in general, a shift of enzymes towards higher optimum temperatures is accompanied by an increase in hydrophobicity and internal packing, mainly through the substitutions G--A, S--A, S--T, and K-*R. Here we compared the very stable Thermus xylose isomerase with the already impressively stable Actinomycetes xylose isomerases. When amino acid substitutions characteristic for Thermus xylose isomerase

erase

are scored by comparison with Actinomycetes xylose isomerases, similar substitutions that may increase the hydrophobicity and internal packing, depending on the position in the structure, are found, namely, G-+A, K-*R, I-+L--*Y, and I+T-*V. In addition, there are G--P substitutions that make the backbone more rigid (20). These substitutions most likely affect thermo-unfolding. There is also frequent substitution of aspartic acid and glutamine. Klibanov et al. (1, 34) showed that these amino acids often react during the irreversible thermoinactivation of Actinomycetes xylose isomerase and other thermostable proteins. The main reactions were deamidation of glutamine and asparagine residues and hydrolysis of the peptide bonding at the carboxy side of aspartic acid. The most frequent substitution between Actinomycetes and Thermus xylose isornerase, D-+E, thus is likely to affect the hydrolysis occurring at higher temperatures, while the substitutions of

glutamine, e.g.,. by histidine, probably influence the other important inactivating reaction, deamidation. It should be noted that D-+E, Q-+H, and G-+P substitutions are much more prominent in our comparison of thermostable xylose isomerases with the more thermostable Thermus xylose isomerase than in the mesophilic to thermophilic comparison described by Argos et al. (3, 21). Thus, the increased thermostability of Thermus isomerase seems to depend on a

more stable structure and a remarkable number of substitutions that might affect irreversible thermoinactivation. Volkin and Klibanov (34) pointed out that xylose isomerase is probably also chemoinactivated by reactions with compounds in high-fructose corn syrup. It is not known which amino acids might be involved. Assuming that inactivation is the sum of thermoinactivation and chemoinactivation, then the latter process might be easier to study in Thermus xylose isomerase since thermoinactivation is much slower. Because the T. thermophilus gene has been cloned, it is now possible to obtain in a simple way large amounts of relatively pure xylose isomerase, e.g., by expression in E. coli and heat treatment of the cell extract. Thus, cloning makes industrial application more feasible and at the same time makes it possible to study several remarkable amino acid substitutions of this very stable xylose isomerase by

mutagenesis (31). Another interesting aspect of the Thermus gene is that conspicuous differences from other xylose isomerases (marked with a circle in Fig. 2, e.g., G137P) exist in regions with substrate binding residues and catalytic residues, regions where homology is relatively high. This confirms the taxonomic conclusion that T. thermophilus diverged long

ago from other eubacteria. T. thermophilus is not necessarily the most thermophilic microorganism that makes xylose isomerase. It will be interesting to see if a xylose isomerase gene from thermophilic archaebacteria (4) can be cloned and to determine what kind of amino acid substitutions can be found in these

organisms. REFERENCES 1. Ahern, T. J., and A. M. Kflbanov. 1985. The mechanism of

TABLE 3. Xylose isomerase activity in heat-treated cell extract of E. coli JM109 harboring the Thermus xylose isomerase gene

Species

Isomerase activity (U/mg of protein)

Isomerase yield (U/liter of culture)

T. thermophilus E. coli E. coli/pUS12XI

6.5 0.1 3.9

10 10 450

irreversible enzyme inactivation at 100°C. Science 228:12801284. 2. Amore, R., and C. P. Hollenberg. 1989. Xylose isomerase from Actinoplanes missouriensis: primary structure of the gene and protein. Nucleic Acids Res. 17:7515. 3. Argos, P., M. G. Rosmann, U. G. Grau, H. Zuber, G. Frank, and J. D. Tratachn. 1979. Thermal stability and protein structure.

Biochemistry 18:5698-5703.

4. Bragger, J. M., R. M. Daniel, T. Coolbear, and H. W. Morgan.

VOL. 173, 1991

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