317

BIOCHEMICAL STUDIES ON AN EXTREME THERMOPHILE The1'TflUs thermophiZus THERMAL STABILITIES OF CELL CONSTITUENTS AND A BACTERIOPHAGE Tairo OSHIMA, Yoshiyuki SAKAKI, Nobuyuki WAKAYAMA, Kimitsuna WATANABE, Ziro OHASHI* and Susumu NISHIMURA* Mitsubishi-Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan and * National Cancer Center Research Institute, Tsukiji, Tokyo, Japan

INTRODUCTION Mechanism of thermophily has been the object of biochemical interest for many years, but has not been clearly answered at the molecular level. Most of the studies have been done using a moderate thermophile, BaciZZus ste=othermophiZus which grows optimally at 60-6S o C. Studies on more rigorously thermophilic bacterium in comparison with the moderate thermophile and mesophile will be one of the most fruitful way to the understanding of thermophily. Thus we have been studying the thermal stabilities of cellular components and bacteriophage of an extremely thermophilic bacterium, The1'TflUs thermophiZus (formerly named Flavobacteriwn thermophiZwn) HB8 which was isolated from a Japanese thermal spa l ,2. Biopolymers and cell organella such as enzymes in glycolytic ° proteln ° synt h eS1S ° 9 , DNA 10 , rl°b osomes 1,9 , pat h 3 - 8 ,enzymes ln tRNA l1 ,12, membrane and membrane lipid 2 ,13, and bacteriophage 14 of the thermophile were studied. These cell components were resistant to heat without exception. We found that the mechanisms of thermal resistance of these biopolymers and organella could be classified into three types: (1) intrinsic stability, that is, a molecule is intrinsically resistant to heat without any specific protector and the stability is caused directly by the chemical structure coded in its gene, (2) stability supported by a ligand of small molecule, namely a cell component is resistant to heat only because of the presence of specific protector and, (3) heat induced stability, heat

H. Zuber (ed.), Enzymes and Proteins from Thermophilic Microorganisms Structure and Function © Springer Basel AG 1976

T . OSHIMA et aZ.

318

stability of a cell constituent is positively correlated to the growth temperature changing its chemical structure depending on the environmental temperature. Thermal stability of any biopolymer, cell organelle or bacteriophage of T. thermophiZus hitherto studied could be interpreted by one of these or, in many cases, by the combination of two or three of these mechanisms. In this communication, we will report some typical examples of these three mechanisms on thermal stability. Intrinsic stability; thermostability of enzymes A number of investigations 3 - 8 on enzymes from T.thermophiZus have shown that the enzyme proteins are, in many cases, stable to heat without the presence of specific cofactors, as reported for many enzymes from other thermophilic sources lS . For instance, glyceraldehyde-3-phosphate dehydrogenase of T.thermophiZus was found to possess remarkable heat stability as shown in Fig. 116. No significant difference was observed between heat stability of the crystalline preparation (Fig. 1) and that of the enzyme in a crude extract, indicating the absence of specific protector for the thermophile glyceraldehyde-3-phosphate dehydrogenase in the cell. The apoenzyme prepared by treating the holoenzyme with charcoal was stable to heat. Thus unusual thermostability of the thermophilic enzyme arises from protein structure itself. Molecular mechanism of the thermostability is still speculative. Fig. 1. Heat stabilities of glyceraldehyde-3-phosphate dehydrogenase from mesophilic source (pig muscle) (a) and T.thermophiZus (b). Solution of crystalline enzyme was heated for 5 min at various temperatures and the remaining activity was assayed at 30°C.

100 80 ;;-

>-

..... :> ..... u «

60 40 20 0

30

BIOCHEMICAL STUDIES ON

319

T. thermophUus

Fig. 2. Comparison of amino acid compositions of glyceraldehyde-3phosphate dehydrogenase from E.coZi

(----), B.stearothermophiZus (_._.), and T. thermophUus (--).

_ Glu . Gln

The comparative studies on the thermophilic enzyme with the mesophilic counterpart revealed similarities in catalytic properties such as pH-activity profile, behavior to SH reagents, subunit structure and specificity, and dissimilarity in stability to heat and denaturating agents such as organic solvents, detergents and urea, suggesting that the structure around the active site of the thermophilic enzyme is similar to that of the mesophilic dehydrogenase, and the active site structure has been preserved during the evolutionary process to thermophile while the other parts of the molecule are modified to make the enzyme thermostable. It was also shown that there is no special features such as covalent cross linkage or the presence of unusual amino acid residues in the thermophile enzyme protein. Studies on the stability of the enzyme perturbed by denaturating agents suggested that the thermostability arises from subtle difference in the architecture of the molecule 16 ,1? This assumption was also supported by the numerical simulation of the denaturation l ? and by comparison of amino acid composition of the thermophile dehydrogenase with those of a moderate thermophile and mesophile enzymes. In Fig. 2, molar contents of amino acid of the enzymes from various sources are illustrated. Although contents of some amino acids such as Arg, Ala and Leu are positively, and some such as Asp and Lys are negatively correlated to the denaturation temperature, generally speaking there is no remarkable difference in amino acid composition. Similarly no significant difference was found in comparing average hydrophobicity defined by Bigelow 18 , number of amino acid residues which may participate in hydrogen

T . OSHIMA et al. Table 1.

320

Comparison of average hydrophobicity and number of amino acid residues which may participate in hydrogen bonding or electrostatic interactions of glyceraldehyde-3-phosphate dehydrogenase from various sources. Lys + His + Arg + Asp + Glu + Ser + Thr + Cys + Met + Thr, per subunit

+ Asp

4.32KJ

152

57

44

4.45

165

65

46

4.20

173

67

44

H~

per residue

T.thermophilus B.stearothermophilus E.eoli

Glu

Lys

+ His + Arg

per subunit

bonding, and number of residues which may be positively or negatively charged and thus participate in electrostatic interactions as given in Table 1. Data in Table 1, do not deny the contributions of hydrophobic, hydrogen bonded or electrostatic forces to the thermostability, but suggest that the increment of these bonding in the thermophile enzyme, if any, must be quantitatively small. The structural difference in relation to the intrinsic stability of the thermophilic enzyme could be considered so small that the exact three dimensional structure of the thermophilic and mesophilic enzymes would be necessary to interpret the difference in stability at the molecular level. Search for explanation of the heat lability of mesophilic enzymes ironically would help the understanding of the abnormal stability of enzymes from thermophilic sources as well. Stability supported by a protector; stability of a phage Some enzymes have been reported to contain factors which increase their thermal stability. For instance, some enzymes from moderate thermophiles such as thermolysin 19 , alkaline protease from thermophilic Streptomyces 20 , a-amylases from B.stearothermophilus 2l , 22 and from an unspecified thermophile, strain V-223, were stabilized by the presence of calcium ion. a-Amylase from V-2 was shown to be more heat labile upon removal of tightly bound calcium ion than calcium free a-amylase from a mesophilic organism. Here we would like to present another example in which the presence of metal ion or other small molecule plays a crucial role for the thermostability. The example is thermophilic bacteriophage (~)YS40

321

BIOCHEMICAL STUDIES ON T. thermophiZus

100,-

-

- - --

- - - --

---...

Fig . 3. Growth of ~YS40 at various temperatures. ~YS40 was grown in HB8 broth l ,2 at the indicated temperature for 5 hr. Burst size means the number of progeny phages per infected cell. A bacteriophage ($YS40) infectious to T. thermophil-us HB8 was isolated and characterized 14 . This is the first phage of extreme thermophiles . Phage YS40 grows over the 80 10 60 :10 temperature range of 56 to Temperature for PhageGrowth(OC) 78°C, and the optimum growth temperature is 65°C (Fig. 3). The phage has a hexagonal head, a tail, a base plate and tail fibers. The phage was easily inactivated by heating at 80°C in a buffer containing lOmM Tris (pH 7.5) and 10 mM MgCl Z (The half life is about 4 min). Also, the phage was sensitive to high concentrations of salt such as NaCl or CsCl (Fig. 4). Electronmicroscopic observation suggested that the head structure was destroyed by these treatments.

60

Fig. 4. Salt sensitivity of $YS40. The phage was suspended in 10mM Tris-HCI buffer, pH 7.5, containing 10mM MgC12 and NaCI at the indicated concentration, and heated at 70°C for 30 min.

o

0.1

0.5

1

Nact Concentration (M)

T . OSHIMA et al.

Fig. S. Elution profile of the stabilizing factor in the hot spring water. The water of hot spring was concentrated 100 fold by evaporation and applied on a Sephadex G-1S column (2 x SS cm). The factor was eluted with 10mM Tris (pH 7.5) containing 10mM MgC12. ~YS40 was added to each fraction and heated at 80°C for 30 min. ni

5

.~

The thermostability of the phage was restored by the addition of 77°C, solid at T < 48°C and intermediate phase at 77°C < T < 48°C. Phase separation 27 (coexistence of both liquid and ()l solid phase) seems to take place over the intermediate region. Similar results were obtained with cells grown at 15 2.5 3.0 other temperatures. The results 1DDDIT are summarized in Table Z. It is noted that the growth temperature is close to the higher inflexion temperature with each sample. In other words, the membrane is adapted to the environmental temperature so that it can be in an appropriate physical state at the specific temperature. 0

.e

0.5

c-

r

tRNA : A model for thermostable biopolymers tRNAs from T.themzophiZus were more stable to heat than those from B.stearothemzophiZus and E.coZi l ,3. The thermostable tRNA is equivalent

Table 2.

Inflexion points of log n - liT relations.

Growth temperature

Inflexion I

Inflexion II

50°C

27°C

53°C

55

44

60

60

43

69

75

48

77

88

52

80

BIOCHEMICAL STUDIES ON

T.the1'lTlophiZus

325

to that of E.aoZi in its function in protein biosynthesis 3 ,9. The thermophile tRNA would be a good model compound for elucidation of the mechanism of thermal stability, because (i) the molecule is small enough to easily determine its primary sequence, (ii) when the primary structure is determined, its three dimensional structure can be speculated based on the recent knowledge obtained by X-ray analysis 28 on yeast tRNA Phe , and (iii) it will be easy to identify the structural difference which relates to the thermostability of the thermophile tRNA since only a small part of the molecule may be modified to make tRNA thermostable without the loss of its biological functions. Structures around the sites interacting with mRNA, rRNA, ribosomal proteins, elongation factors, and aminoacyltRNA synthetase should be preserved to retain its role in protein synthesis during adaptation to high temperature. Formylmethionine specific tRNA (tRNA~et) was extracted from T. the1'lTlophiZuB cells, purified and its nucleotide sequence was studied. By analyzing the nucleotide sequences of fragments obtained by RNase Tl and RNase A digestions, the primary structure was proposed as illustrated in Fig. 7, in which a clover-leaf model of the thermophile tRNA~et is Au 77 shown. Six bases were C C different from the structure A pC A of E.aoZi tRNA~et; 2'-0-methylG·C C·G guanosine at position 19 G·C G·C instead of guanosine in E.aoZi G·C 60 U tRNA, cytosine at 51 instead C CGC CC ArrlA A of uridine, guanosine at 52 CCGA CGAG G gCGGG s '" C instead of C, 5-methyl-2U U Gm GCUC ni'G thiouridine (m 5s 2U) at 55 G AG 2OGOA instead of ribothymidine (T), U·A C·G I-methyladenosine at 59 instead G•C S=rrPs2U G·C of A, and C at 64 instead of G. G· C40 Cm A The G-C pair content in U A base-paired regions of the C A U thermophile tRNA~et is estimated to be 90% and the Fig. 7. Proposed primary sequence of T.the1'lTlophiZus tRNA~t. high G-C pair content may, at

stS·

T . OSHIMA et at.

326

T .Ih«m:>phil us o mel f

90

Fig. 8. Relation between G-C pair content and melting temperature of tRNA.

60 ...E

70

50

60

70

60 G- C

90

100

Pai r 'f,

least partly, be responsible for the high melting temperature of the tRNA (89°C) . It was found that circular dichroic (CD) difference spectra of tRNA upon heat denaturation correlated with G-C pair content ll . ~ased on this observation, the CD difference spectrum study sugges ted that tRNA from T. thermophiZuB contains more G-C pairs than those from E.coZi and average G-C pair content in the thermophile tRNA is 85-90%. As an average, three to four A-U pairs per molecule would be replaced by G-C pairs in tRNA from T.thermophiZuB, thus increment of three or four hydrogen bonds would be expected inside of the thermophile tRNA molecule. We have found that the high melting temperature of tRNAs from T.therrnophiZuB could not be explained fully by the increment of G-C pairs as shown in Fig. 8. A linear relation was observed between G- C pair content and melting temperature of tRNA from E.coZi, but tRNAmixture and tRNA~et from the extreme thermophile did not fallon the straight line in Fig. 8. There must be some other mechanism(s) responsible for the thermostability than the G-C pair content. One possible candidate is the presence of modified base(s). Among the minor components, mlA and mSs 2U are not found in E.coZi tRNA. It is, however, unlikely that mlA has a crucial role in the thermostability of the thermophile tRNA, since mlA is known to be present widely in eukaryotic tRNAs which do not have any unusual thermal stability. On the other hand, the presence of mSs 2U in the primary sequence of the thermophile tRNA is unique feature l2 • The view that mSs 2u may play an important role in the thermostability was supported by measurements of CD bands in near-ultraviolet region

327

BIOCHEMICAL STUDIES ON T. thermophilus

(300-400 nm). The CD bands in this region are correlated to the conformations of such minor components as s 4u or m5s 2U. A positive band *at around 310 nm was assigned to m5s 2u, because this band did not disappear when s 4U base was chemically converted to U. As temperature of the thermophile tRNAwet solution was raised, the main band centered at 265 nm gradually decreased suggesting partial loss of the secondary structure of the tRNA, but band at 310 nm scarcely changed until up to 86°C, and then it rapidly decreased as shown in Fig. 9. The profile of the change in the band at 310 nm upon heating was close to the melting profile measuring the change in absorbancy at 260 nm. This observation suggests a close relation between heat denaturation of the tRNA and the conformation of m5s 2U base.

25°

300

350

400

WAVELENGTH (nm)

Fig. 9. Circular dichroic bands of T.thermophilus tRNA in near ultraviolet region at various temperatures. The spectra were recorded on a JASCO J-20 Spectropolarimeter connecting a circulating thermostatic bath.

Fig . 10. A proposed three dimensional structure of

T.thermophilus

tRNA~et.

Four nucleotide residues were superimposed on the structure reported 28 for yeast tRNAPhe.

T .OSHIMA et at. Table 3.

328

Effect of magnesium ion on melting temperature of tRNA. After tRNA was treated with sodium CyDTA to remove tightly bound Mg, melting temperature was measured in 10mM Tris buffer (pH 7.5) containing the indicated concentration of Mg++. Mg++

tRNA

0

concentration(M)

10- 4

10- 2

G-C pair content(%)

E. coli tRNAMet m

53

76

77

65

E.coli tRNA~et

55

81

83

87.5

T.thermophiZu8 tRNAMet f

54

87

89

90

Z-Thiouridine derivatives are known to strengthen the stacking interaction with the neighboring bases Z9 ,30. In three dimensional structure assumed for the thermophile tRNA~et based on the data reported Z8 for yeast tRNAPhe (shown in Fig. 10), m5s ZU at position 55 which is base-paired with mI A-59, stacks on the neighboring base Gm-19 thus T~C loop is connected with DHU loop. It is conceivable that the stacking force between m5s ZU and Gm is stronger than that between T and G in mesophile tRNA and thus T~C loop is connected more tightly to DHU loop in the thermophile tRNA resulting in high stability of maintenance of the folded structure of the tRNA. Another mechanism which is also responsible for the thermostabi1ity of the thermophile tRNA is the binding of magnesium ion. As reported for mesophile tRNA, the stability of tRNA from the thermophile was also greatly affected by the concentration of magnesium. As shown in Table 3, the remarkable thermostability of tRNA from the thermophile was observed only in the presence of magnesium indicating the crucial role of magnesium in the stability of tRNA molecule. Finally we found that the thermal stability of tRNA from T. thermophiZus is dependent on the growth temperature. Unfractionated tRNA was isolated from cells grown at various temperatures ranging from 50° to 80°C. The melting temperature was correlated positively with the growth temperature as shown in Fig. 11. Nucleotide analysis revealed that contents of only two components, m5s ZU and T, varied upon changing the growth temperature. The sum of the mole fraction of m5s ZU plus that of T was constant. A linear relation was observed

329

BIOCHEMICAL STUDIES ON T.thermophilus

Tm

(Oe)

90

~;.

--

Fig. 11. The relationship between mole fraction of 5-methyl-2-thiouridine residue and melting temperature of tRNA. S 5-methyl-2-thiouridine, T = ribothymidine.

80

between mole fraction of m5 s 2U and the melting temperature of tRNA preparation as shown in ~-- --Fig. 11. This observation also 70 strongly supports the idea that the presence of'm 5s 2U is directly 0.5 1 5= 0 related to the thermostability T= 1 0.5 o of the thermophile tRNA. The results stated here indicate that the modification of T to m5s 2U (thiolation) is dependent on the environmental temperature resulting in the change of melting temperature of the tRNA. The thermos tab i l i ty of T. thermophilus tRNA is explained by the combination of three mechanisms for the thermal resistance; (i) intrinsic stability arised by the increment of G-C pair content and by tight stacking between m5 s 2U-55 and Gm-19, (ii) protection by magnesium ion, and (iii) temperature induced stability by thiolation of T base to m5 s 2U base depending upon the environmental temeprature.

---

REFERENCES l.

2. 3.

Oshima, T. Oshima, T. 102-112. Oshima, T. (Rohlfing, Publishing

& Imahori, K. (1971) & Imahori, K. (1974)

J. Gen. Appl. Microbiol. 17, 513-517. Intern. J. Syst. Bacteriol. 24,

(1972) in Molecular Evolution : Frebiological and Biological D.L. and Oparin, A. I. eds) P.399-423, Plenum Co. , New York.

T . OSHIMA et al.

4.

Yoshizaki, F., Oshima, T. & Imahori, K.

(1971)

J.

Biochem. 69,

1083-1089 5.

Yoshida, M., Oshima, T. & Imahori, K. Commun. 43,

6.

(1971) Biochem. Biophys. Res.

36-39.

Yoshida, M., Oshima, T. & Imahori, K.

(1973) J. Biochem. 74,

1183-1191. 7.

Yoshida, M.

(1972)

8.

Yoshida, M. & Oshima, T.

1087-1093.

Biochemistry 11, (1971)

Biochem. Biophys. Res. Commun. 45,

495-500. 9.

Ohno-Iwashita, Y., Oshima, T. & Imahori, K. Mik:r>obiol. 15,

(1975)

Z. AUg.

131-134.

10.

Oshima, T. & Imahori, K.

11.

Watanabe, K., Seno, T., Nishimura, S., Oshima, T.

12.

( 19 7 3 ) Po lymer J. 4, 539 - 5 5 2 . Watanabe, K., Oshima, T., Saneyoshi, M. & Nishimura, S. FEBS Letters 43,

(1974)

Biochem. 75, 179-183.

J.

& Imahori, K. (1974)

59- 63.

13.

Oshima, M. & Yamakawa, T.

14.

Sakaki, Y. & Oshima, T.

15.

Singleton, Jr., R. & Ame1unxen, R.E.

(1974)

(1975)

Biochemistry 13, 1140-1146. J.

1449-1453.

Virology 15, (1973)

Bacteriol. Rev. 37,

320-342.

& Imahori,

16.

Fujita, S.C., Oshima, T.

17.

Fuj ita, S. C. & Imahori, K., in

K., submitted

Pep tides, Polypeptides & Proteins

& Lotan,

(B1out, E.R., Bovey, F.A., Goodman, M. 229, John Wiley

& Sons,

N. eds) P.217-

London and New York.

18.

Bigelow, C.C. (1967)

19.

Endo, S. (1962)

20.

Mizusawa, K., Ichishima, E. & Yoshida F.

(1964)

21.

884- 895. Pfue11er, S.L. & Elliott, W.H.

Biol. Chem. 244, 48-54.

22.

Ogasahara, K., Imanishi, A. & Isemura T.

J. Theoret. Biol. 16,

187- 211.

Bakko Kogaku Zasshi 40, 346-353. Agr. Biol. Chem.

28,

(1969)

J.

(1970)

J. Biochem. 67,

77-82. 23.

Hasegawa, A., Miwa, N., Oshima, T. & Imahori, K. J. Biochem.

24.

Oshima, M. & Miyagawa, A.

25.

Shinitzky, M., Dianoux, A.C., Git1er C. & Weber, G.

in press

Biochemistry 10, 26.

(1974)

Lipids 9, 476-480.

Cogan, U., Shinitzky, M., Weber, G. & Nishida, T. Biochemistry 12,

(1971)

2106 - 2113 . 521- 528.

(1973)

330

331

BIOCHEMICAL STUDIES ON T. thermophilus

27.

Linden, C.D., Wright K.L., McConnell, H.M. & Fox, C.F. Proc. Nat. Acad. Sci. USA 70,

28.

Kim, S.H., Suddath, F.L., Quigley, G.J., McPherson, A., Sussman, J.L., Wang, A.H.J., Seeman N.C. & Rich, A. Science 185,

29.

(1974)

435 - 440.

Mazumdar, S.K., Saenger, W. & Scheit, K.H. 85,

30.

(1973)

2271-2275.

(1974)

J. Mol. Biol.

213-229.

Bahr, W., Faerber, P. & Scheit, K.H. 535-544.

(1973)

Eur. J. Biochem. 33,