THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. 265, No. 12, Issue of April 25, pp. 6931-6935, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Nonprotein Amino Acid Furanomycin, Unlike Isoleucine in Chemical Structure, Is Charged to Isoleucine tRNA by Isoleucyl-tRNA Synthetase and Incorporated into Protein*

(Received for publication, October 10, 1989)

Toshiyuki KohnoS, Daisuke KohdaS$, Mitsuru HarukiS, Shigeyuki YokoyamaSn, and Tatsuo Miyazawall From the SDepartment of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 and the 11 Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama, 240, Japan

Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine. The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the trans- ferred nuclear Overhauser effect method. The confor- mation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of fur- anomycin is unlike that of L-isoleucine. By E. coli IleRS, E. coli tRNA”” was charged with furanomycin as efficiently as with L-isoleucine. Furthermore, fur- anomycyl-tRNA”” was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNA”‘. Furanomycin was found to be incorporated into j3- lactamase precursor by in vitro protein biosynthesis. A newly designed amino acid will probably be incor- porated into proteins, provided that the new amino acid takes a similar conformation as a protein-consti- tuting amino acid in the active site of an aminoacyl- tRNA synthetase.

In the translation process of protein biosynthesis, 20 amino acids are incorporated into proteins. Each of those protein- constituting amino acids is distinguished strictly from the other 19 amino acids and charged to tRNA by the cognate aminoacyl-tRNA synthetase. However, substrate recognition of aminoacyl-tRNA synthetases is not necessarily strict in discriminating against nonprotein amino acids. For example, L-norleucine is activated by methionyl-tRNA synthetase and incorporated in place of L-methionine in in vivo and in vitro biosynthesis (1, 2), and canavanine is activated by arginyl- tRNA synthetase and incorporated into proteins (3,4). Indeed each of these amino acids is similar to a protein-constituting amino acid in chemical structure.

Nonprotein amino acid furanomycin (Fig. IA) has been found as an antibiotic from Streptomyces L-803 (5). The side chain of furanomycin has a five-membered heterocyclic ring with a double bond. Toxicity of furanomycin is not found in

* This work was supported in part by Grants-in-Aid 59222005 and 60060004 for Scientific Research and Distinguished Research, re- spectively, from the Ministry of Education, Science and Culture of Japan. 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 USC. Section 1734 solely to indicate this fact.

f Present address: Dept. of Medical Chemistry, Tokyo Metropoli- tan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan.

B TO whom correspondence should be addressed.

the presence of natural amino acids, in particular L-isoleucine (5). Furthermore, furanomycin has been found to inhibit isoleucyl-tRNA formation (6). In this study, we have found that furanomycin is bound to Escherichia coli isoleucyl-tRNA synthetase (IleRS).’ In the active site of IleRS, furanomycin takes a conformation similar to that of the natural substrate L-isoleucine. Furanomycin is efficiently charged to E. coli isoleucine tRNA and incorporated into protein.

EXPERIMENTAL PROCEDURES

Materials-Furanomycin was provided by Dr. Terui of Shionogi Co. Ltd. E. coli tRNA”” was prepared as described (7). IleRS was purified from E. coli cells harboring i/-es gene on a plasmid (8). Polypeptide chain elongation factor Tu (EF-Tu) and EF-Tu.EF-Ts were purified from crude extract of Thermus thermophilus HB8 cells by successive chromatography on columns of DEAE-Sephadex A-50, DEAE-Toyopearl, and butyl-Toyopearl. EF-Tu.EF-Ts was finally purified by high performance liquid chromatography on a gel filtration column of G3000SW. L-Isoleucine, L-valine, and L-leucine were pur- chased from Wako Pure Chemical Industries (Tokyo). 2-(p-Tolui- dino)naphthalene-6-sulfonate (TNS) was from Nakarai Chemicals (Tokyo). ‘Hz0 (99.85%) was purchased from the Commissariat a 1’Energie Atomique. L-[‘?]Isoleucine, L-[35S]methionine, and [3H] GDP were purchased from Amersham Corp.

Determination of Dissociation Constant of Complex of ZleRS and Amino Acid-The dissociation constant of a complex of IleRS and amino acid at 37 “C was determined by the fluorescence analysis of IleRS-bound TNS. The fluorescence of TNS was excited at 366 nm and observed at 470 nm by the use of a Hitachi Fluorescence Spec- trometer F-4000. The sample solutions (pH 7.0) contained 10 mM potassium phosphate, 5 mM 2-mercaptoethanol, 50 mM KCl, 0.2 pM IleRS, 20 ELM TNS, and amino acid at various concentrations. The dissociation constant was determined from the dependence of the fluorescence intensity on the concentration of amino acid (9, 10).

NMR Measurements-The ‘H,O solution of IleRS for NMR meas- urements was prepared as described previously (10). The sample solution (0.35 ml) contained 10 mM potassium phosphate buffer (pH 7.0), 50 mM KCl, 0.25 mM IleRS, and 6.3 mM furanomycin. The 400- MHz ‘H NMR spectra were recorded on Bruker AM-400 and WM- 400 spectrometers (10); a total of 512 transients (each for 1.6 s) was accumulated and a line broadening of 1 Hz was applied. The amino- acylation activity of IleRS was assayed before and after the NMR measurements; no loss of the activity was found.

Transferred Nuclear Overhauser Effect (TRNOE)-For each proton of IleRS-bound furanomycin, the TRNOE action spectrum (the de- pendence of resonance intensity on the frequency of irradiation at the interval of 0.05 ppm) was obtained as described (10). The effect of spin diffusion (from the protein globule of IleRS to furanomycin) was largely eliminated by taking I(t)/I,(t), where I,(t) is the intensity of ligand resonance with a control irradiation (at 8.4 ppm) of the protein globule (11). Time-dependent TRNOE on the proton reso-

’ The abbreviations used are: IleRS, isoleucyl-tRNA synthetase; EF-Ts, polypeptide chain elongation factor Ts; EF-Tu, polypeptide chain elongation factor Tu; TNS, 2-(p-toluidino)naphthalene- 6-sulfonate; TRNOE, transferred nuclear Overhauser effect.

6931

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6932 Furanomycin Is Charged to tRNA by Isoleucyl-tRNA Synthetase

A a

NHSHCOOH NHzCHCOOH CH

C/Hz’CHs CH3

CH3

FIG. 1. Chemical structures of (A) L-isoleucine and (B) fur- anomycin.

nances of IleRS-bound furanomycin was measured as described pre- viously (10).

Arn&oacylation of tRNA-Aminoacylation was performed at 37 “C in a reaction mixture (oH 7.5) containine 100 mM Tris-HCl buffer. 5 mM magnesium acetate, 10 &IM KCl, c mM ATP (mixture A), ‘in addition to IleRS, tRNA”‘, and amino acid. As for the aminoacylation for 5 min at various concentrations of amino acid, the reaction mixtures (100 ~1) contained 1.5 KM IleRS and 1.7 KM tRNA”‘. As for the time course of aminoacylation, the reaction mixtures (10 ~1) contained 0.05 UM IleRS, 20 UM tRNA”‘, and 1 mM amino acid. After incubation, the’ reaction’was stopped by addition of 0.5 volume of 2 M potassium acetate buffer (pH 4.0) to the reaction mixture. The 01- amino group of aminoacyl-tRNA”’ was acetylated by the reaction with 0.66 volume of acetic anhydride. Acetylaminoacyl-tRNA (and uncharged tRNA) was precipitated by addition of 0.5 volume of 0.6 M NaCl and 3 volumes of ethanol. After drying, uncharged tRNA*” was assayed by the use of 10 pM L-[%]isoleucine in mixture A.

Determination of Binding Constant of Ternary Complex-Amino- acylation reaction was performed at 37 “C for 6 min in a reaction mixture (pH 7.8, 0.5 ml) containing 100 mM Tris-HCl, 10 mM mag- nesium acetate, 10 mM KCl, 2 mM ATP, 1.25 pM IleRS, 17 pM tRNA”“, and 100 pM amino acid. The reaction was stopped by addition of 7 ~1 of 2 M acetic acid. The aminoacyl-tRNA was extracted with an equal volume of phenol and then precipitated with 2.5 volumes of ethanol. The precipitate was dissolved in 1 ml of 50 mM Tris-HCl buffer (pH 7.9) containing 10 mM magnesium acetate and 5 mM 2- mercaptoethanol. The concentration of tRNA was determined from the absorbance at 260 nm.

The binding constant of the ternary complex of EF-Tu. GTP and aminoacyl-tRNA was determined by the nitrocellulose membrane filter method (12). Ligand exchange of EF-Tu and complex formation was performed at 30 “C for 30 min in a reaction mixture (pH 7.9, 10 ~1) containing 50 mM Tris-HCl, 10 mM magnesium acetate, 5 mM 2- mercaptoethanol, 0.4 FM EF-Tu.GDP, 14 nM EF-Ts, 100 FM GTP, 5 FM [3H]GDP, and aminoacyl-tRNA at various concentrations. After incubation, the reaction mixture was filtered through a nitrocellulose membrane, and the membrane was washed twice with 50 mM Tris- HCl buffer (pH 7.9) containing 10 mM magnesium acetate and 5 mM 2-mercaptoethanol. The binding constant of the ternary complex was obtained from the radioactivity of EF-Tu. [3H]GDP bound to the membrane filter.

In Vitro Protein Synthesis-The reaction mixture (mixture B) contained, in a volume of 7.5 ~1, 83 pg/ml plasmid pAT153 as the template, 1.3 @M L-[%]methionine, and the other protein-constitut- ing amino acids (each at about 200 pM) except L-isoleucine, in addition to 1.25 ~1 of E. coli S-30 extract and 1.9 ~1 of supplement solution from Amersham’s nrokarvotic DNA directed translation kit. Mixture B does not include L-iskleucine and is not sufficient for efficient protein biosynthesis. The reaction mixture was divided into three parts. To the first part, L-isoleucine was added to a concentra- tion of 200 FM. To the second part, nothing was added. To the third part, furanomycin was added to a concentration of 10 mM. Each part was then incubated for 1 h. Then the L-methionine chase solution was added to each part. This allowed complete synthesis of protein chains that were prematurely terminated because of a low concentra- tion of L-[“Slmethionine. Each part was finally subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorographic ex- posure (by the use of Amersham Amplify).

RESULTS

Dissociation Constants of Complexes of IleRS and Amino Acids-The fluorescence intensity of IleRS . TNS was reduced upon binding of amino acid; the maximum quenching was 10.8 and 7.1%, respectively, upon binding of L-isoleucine and furanomycin. The dependence of fluorescence intensity upon amino acid concentration was plotted as described (13). The

dissociation constant of the complex of IleRS and amino acid at 37 “C was obtained as 0.010 mM for L-isoleucine, 0.031 mM

for furanomycin, 0.73 mM for L-valine, and 1.6 mM for L- leucine. Thus, the binding of nonsubstrate amino acid, L-

valine or L-leucine, is much weaker than that of the substrate L-isoleucine. In contrast, the binding of nonprotein amino acid furanomycin is nearly as strong as L-isoleucine.

NMR Spectra of Furanomycin-The 400-MHz NMR spec- trum of free furanomycin is shown in Fig. 2A, where the proton resonances have been assigned by the double resonance method. This preparation of furanomycin was highly pure, so that the proton resonances of isoleucine or other amino acids were not observed at all in the spectrum (Fig. 2A) (the signal- to-noise ratio for the methyl proton resonance of furanomycin was higher than 106). The NMR spectrum of furanomycin (6.3 mM) in the presence of E. coli IleRS (0.25 mM) is shown in Fig. 2B. In this solution, 99.5% of IleRS is in the complex with furanomycin, as estimated from the dissociation constant of 0.031 mM. The conformation of furanomycin bound to IleRS was elucidated by the analysis of intramolecular TRNOE under the fast exchange condition as described (10). TRNOE action spectra of furanomycin in the presence of IleRS are shown in Fig. 3.

In the action spectrum for H, (Fig. 3A), sharp minima were observed at 5.45 and at 5.90 ppm, which correspond to the Ho proton and the H+ proton resonances, respectively. Con- versely, in the action spectra for HP (Fig. 3B) and H?P (Fig. 3C), sharp minima due to the H, proton were observed at 3.90 ppm. Note that these sharp minima are not overlapped by broad minima due to intermolecular TRNOE. This indicates that the TRNOE data for these pairs of protons of furano- mycin are useful for elucidating the conformation of IleRS- bound furanomycin.

Conformation of IleRS-bound Furanomycin-Dependence of the H, proton resonance intensity of furanomycin upon duration of irradiation of other protons is shown in Fig. 4. AS the duration of irradiation of the HB proton was increased from 0.05 to 0.8 s, the intensity of the H, proton resonance was decreased by 19%. Therefore, from the initial slope and

f A .I 62 Y2 ‘9 81

L

FIG. 2. 400-MHz NMR spectra of furanomycin in the ab- sence (A) and in the uresence (B) of E. coli IleRS. The concen-

~ I

tration of furanomycinand IleRS is 6.3 and 0.25 mM in *Hz0 solution (37 “C, pH 7.0). The nomenclature for furanomycin is shown in Fig. 5.

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Furanomycin Is Charged to tRNA by Isoleucyl-tRNA Synthetase 6933 I TABLE I

0.9 t

CHEMICAL SHIFT (ppm)

FIG. 3. TRNOE action spectra. A, H,; B, H,; C, HTz; D, Haz; E, Hai; and F, H, protons of furanomycin (6.3 mM) in the presence of IleRS (0.25 mM) at 37 “C. Relative peak intensities of the proton resonances of furanomycin in the presence of IleRS were obtained from the difference between the NMR spectrum of the mixture of furanomycin and IleRS and that of IIeRS alone. The duration of irradiation was 0.6 s. The proton NMR spectrum of furanomycin in the presence of IleRS, from which the spectrum of IleRS alone has been substracted, is shown (C) for comparison.

01 I I 0 0.2 0.4 0.6 0.6

DURATION OF IRRADIATION (8)

FIG. 4. Time-dependent TRNOE. Dependence, on irradiation time, of the relative resonance intensity [I(t)/IJt)] of the H, proton on irradiation of HB (O), HTz (O), H&2 (X), and Hai (B) protons of furanomycin (6.3 mM) in the presence of IleRS (0.25 mM) at 37 “C. The relative intensities of proton resonances were obtained as de- scribed in the legend to Fig. 3.

the mole fraction of IleRS-bound furanomycin (0.04), the cross-relaxation rate (a) for the proton pair H,-Ho was ob- tained as 45 f 8 s-l. Similarly, the cross-relaxation rate for the pair H,-HY2 was obtained as u’ = 23 & 7 s-‘. u values for other proton pairs are listed in Table I. The cross-relaxation rates for this system may well be treated as being approxi- mately proportional to the inverse sixth power of interproton distances (10).

Because of the ring structure in the side chain of furano- mycin (Fig. l), only three rotamers about N-C, -CB -C,z are possible, namely gauche+, gauche-, and trans forms. For each of these rotamers, the inverse sixth power of interproton distances (rs6) was calculated for the four proton pairs as shown in Table I. Clearly, the cross-relaxation rates for the four proton pairs are proportional to the P values for the

Apparent cross-relaxation rates u’ of ZleRS-bound furanomycin at 37 “C and inverse sixth power of interproton distances for rotamers

about N-C, -C, -CYz oi furanomycin

HI” H2” Gauche+

10-S r-0

Gauche- Trans s-1 nmm6 nmee nme6

45 + 8 0.41 0.41 0.11 23 + 7 0.01 0.21 0.09

l:: E:: a+5 8+5 0.00 0.02 0.01 0.01 0.02 0.08

“On irradiation of proton Hl, the resonance of proton H2 is observed; distance between proton Hl and proton H2.

A B

FIG. 5. Conformations of L-isoleucine (10) (A) and furano- mycin (B) in the IleRS-bound state.

TIME (min)

FIG. 6. Time course of aminoacylation of tRNA by E. coli IleRS. The fraction of tRNAse charged with furanomycin (0) and L- isoleucine (0) are shown against the duration of incubation.

gauche- form rather than the gauche+ form or the trans form. The conformation of the gauche- form of furanomycin is shown in Fig. 5B. This conformation of IleRS-bound furano- mycin is similar to that of IleRS-bound L-isoleucine (10).

Enzyme Activity of IleRS for Furanomycin-Isotope-labeled furanomycin was not available, so the back titration method (14) was used for determination of the fraction of tRNAne charged with furanomycin, L-isoleucine, or L-valine. In this study, aminoacyl-tRNA was effectively stabilized (against hydrolysis) by acetylation of the aminoacyl group (15). Note that the amino acid accepting activity of E. coli tRNA”’ itself was not affected by the treatment with acetic anhydride. Thus, after aminoacylation of tRNAiL by IleRS and acetylation by acetic anhydride, the fraction of uncharged tRNA*” was de- termined by aminoacylation with L- [ “C]isoleucine.

At a low concentration (0.05 PM) of IleRS, the time course of the aminoacylation of tRNA’” was obtained for furano- mycin, which is highly pure and practically free of isoleucine as described above, and for L-isoleucine (Fig. 6). Thus, fur- anomycin was found to be charged to tRNA”‘, although the rate of furanomycyl-tRNA formation was slower than that of isoleucyl-tRNA formation. Furthermore, at a higher concen- tration (1.5 PM) of IleRS as in the cell (16), the aminoacyl- tRNA formation for furanomycin, L-isoleucine, or L-valine was assayed for a concentration range of 0.1-10 PM. As shown in Fig. 7, E. coli tRNAne was charged with furanomycin as efficiently as with the natural substrate L-isoleucine by E. coli IleRS. In contrast, tRNA’” was hardly charged with L-valine,

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6934 Furanomycin Is Charged to tRNA by Isoleucyl-tRNA Synthetase

0’ I 10“ 10’6 10’5 10.4

AMINO ACID (M)

FIG. 7. Aminoacylation of tRNA”’ by E. coli IleRS. The fraction of tRNA”’ charged with furanomycin (O), L-isoleucine (O), and L-valine (X) are shown against the amino acid concentration.

another protein-constituting amino acid. Thus, IleRS was found to catalyze the formation of furanomycyl-tRNA as efficiently as isoleucyl-tRNA under the condition that the enzyme concentration is high enough as in the cell.

Dissociation Constant of Furanomycyl-tRNA . EF-Tu. GTP Complex-The dissociation constant of the complex of ami- noacyl-tRNA and EF-Tu. GTP was determined in two steps. In the first step, the ligand exchange of EF-Tu

EF-Tu. GDP + GTP = EF-Tu.GTP + GDP

was performed at various concentrations of GTP but in the absence of aminoacyl-tRNA. From the concentration of EF- Tu. [“HIGDP bound to a membrane filter, the concentration of free [“H]GDP was obtained and the equilibrium constant was determined as K = 0.026. In the second step, the ternary complex formation

EF-Tu. GTP + aminoacyl-tRNA = aminoacyl-tRNA .EF-Tu. GTP

was performed by addition of various concentrations of ami- noacyl-tRNA to the reaction mixture. From the equilibrium constant K and the concentration of EF-Tu. [3H]GDP, the dissociation constant of aminoacyl-tRNA . EF-Tu . GTP com- plex was determined as 2.2 x 10e7 M for furanomycyl-tRNA”‘. This value is nearly the same as that for isoleucyl-tRNA”” (2.0 x low7 M) and is much smaller than that for uncharged tRNA”’ (10 X 10m7 M)." Thus, furanomycyl-tRNA”’ forms a complex with EF-Tu. GTP as tightly as isoleucyl-tRNA”‘, suggesting that furanomycyl-tRNA”” will be bound to the A site of the ribosome and furanomycin will be incorporated, in place of L-isoleucine, into proteins.

In Vitro Incorporation of Furanomycin into Protein-The autoradiogram of the protein synthesized in vitro with the plasmid pAT153 as the template is shown in Fig. 8. This plasmid has bla gene that codes for p-lactamase with a molec- ular weight of 27,000 and tet gene that codes for a protein (tetracycline-resistant) with a molecular weight of 40,000. With all the protein-constituting amino acids in the reaction mixture (lane A), a major band corresponding to a molecular weight of 30,000 was found. This molecular weight is slightly higher than that of /3-lactamase and corresponds to a precur- sor of this enzyme. This suggests that the precursor was not processed in in vitro protein synthesis. On the other hand, tet gene product (molecular weight of 40,000) was not detected, probably because the promoter of tet gene was much weaker than that of bla gene (17).

The band due to /3-lactamase precursor was hardly detected if the reaction mixture was deficient in L-isoleucine (Fig. 8, lane B). The very weak band due to this precursor is probably due to contamination of L-isoleucine in E. coli S-30 extract.

’ There have been no reports on the membrane filter determination of the dissociation constants for the complexes of 7’. thermophilus EF-Tu with isoleucyl or uncharged tRNA”‘.

ABC

FIG. 8. Protein synthesized in vitro subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and auto- radiography. Lane A, all the protein-constituting amino acids were included in the reaction mixture; lane B, 19 protein-constituting amino acids except L-isoleucine were included in the mixture; and lane C, furanomycin was added to the reaction mixture in addition to 19 protein-constituting amino acids except L-isoleucine. The band due to pre-/3-lactamase is shown with an arrow.

However, if furanomycin is included, in place of L-isoleucine, in the reaction mixture, a major band due to p-lactamase precursor was again detected (Fig. 8, lane C). These results indicate that furanomycin was incorporated, in place of L- isoleucine, into fi-lactamase as efficiently as L-isoleucine, since the contamination of L-isoleucine in this preparation of furanomycin is negligible.

DISCUSSION

Nonprotein amino acid furanomycin (Fig. 1B) has been found to inhibit the enzymatic activity of E. coli IleRS (6). In this study, furanomycin was found to bind with IleRS much more tightly than nonsubstrate amino acids L-valine and L- leucine and nearly as tightly as the natural substrate L- isoleucine.

The affinity of an amino acid to aminoacyl-tRNA synthe- tase is determined by the conformation of enzyme-bound amino acid. Previously, the conformations of L-isoleucine and L-valine bound to the active site of E. coli IleRS were eluci- dated (10). These molecules were found to take the same conformation except that the &methyl group in L-isoleucine was replaced by a hydrogen atom in L-valine. However, bind- ing of L-valine with IleRS is much weaker than that of L- isoleucine; the d-methyl group of L-isoleucine appears to be important in the discrimination of L-isoleucine from nonsub- strate L-valine.

In contrast with L-valine, the binding of furanomycin with IleRS is nearly as tight as L-isoleucine, although the chemical structure of furanomycin is unlike L-isoleucine (Fig. IA). However, the overall conformation of IleRS-bound furano- mycin was found to be similar to that of L-isoleucine (Fig. 5, A and B). Probably the pocket of IleRS provided for the 6- methyl group of L-isoleucine is occupied by the t-methyl group of furanomycin, significantly stabilizing the complex of fur- anomycin and IleRS.

The tight binding of amino acid furanomycin to E. coli IleRS suggests the possibility that E. coli tRNA”’ is charged with furanomycin by the enzyme. In fact, tRNA”’ was found to be charged with furanomycin as efficiently as with the natural substrate L-isoleucine (Fig. 7). This is in sharp con- trast to the case of L-valine. Protein-constituting amino acid L-valine is activated by IleRS to form valyl-AMP, with a k,,,/ & value as small as %SO of that for the formation of isoleucyl- AMP (18). However, valyl-AMP is then hydrolyzed by the enzyme in the proofreading stage (18), so that tRNA’le is not

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Furanomycin Is Charged to tRNA by Isoleucyl-tRNA Synthetase 6935

charged with L-valine. The high efficiency in the aminoacyl- ation of tRNA’le with furanomycin indicates that furanomy- cyl-AMP is not hydrolyzed in the proofreading stage in the presence of tRNA”‘. These results are consistent with the double-sieve model (18, 19); amino acids larger than the cognate one are rejected by the first sieve while amino acids smaller than the cognate one are excluded by the second sieve. Furanomycin takes a conformation which is neither larger nor smaller than the active site pocket of IleRS so that this nonprotein amino acid is admitted through the first and second sieves.

Formation of furanomycyl-tRNA”” suggests that furano- mycin is brought to the A site of the ribosome provided that furanomycyl-tRNA”’ tightly binds with EF-Tu. GTP. In fact, the complex of furanomycyl-tRNA”’ and EF-Tu.GTP was shown to be as tight as the complex of isoleucyl-tRNA”’ and EF-Tu.GTP. This further suggests that furanomycin is in- corporated into proteins upon binding of furanomycyl- tRNA1le with the A site of the ribosome. In fact, furanomycin was finally found to be incorporated, in place of L-isoleucine, into p-lactamase precursor in in vitro protein synthesis (Fig. 8), although the chemical structure of furanomycin is nothing like a protein-constituting amino acid L-isoleucine (Fig. lA).

Thus, furanomycin was now found to be an unnatural substrate of IleRS. Although furanomycin was recognized and activated, in place of L-isoleucine, by the enzyme IleRS, the conformation of furanomycin is not exactly the same as L- isoleucine. We note that the furanomycin side chain is more bulky than that of t-isoleucine, in particular around the Caz atom (Fig. 5B). Upon being substituted for L-isoleucine, the bulky side chain of furanomycin possibly disturbs the native conformations and functions of proteins that are vital to the cell physiology. The perturbation of conformations of those proteins is probably responsible for the cytotoxicity (5) of furanomycin, which has been found as an antibiotic from Streptomyces L-803.

Furanomycin was found to be charged to tRNA”” and incorporated into proteins, although this amino acid is unlike any of the protein-constituting amino acids in chemical struc- ture. Therefore, a nonprotein amino acid with a useful prop- erty may be incorporated into proteins, provided that the amino acid is not discriminated from the natural substrate by an aminoacyl-tRNA synthetase. This encourages us to syn- thesize alloproteins (20) that have novel nonprotein amino acid residues. Already a basic strategy has been established

for preparing alloproteins in vivo and human epidermal growth factor containing a norleucine residue has been syn- thesized by E. coli (20). Such a new technique will drastically expand the possibility of designing specific proteins with novel functions.

Acknowledgments-We are grateful to Dr. A. Terui of Shionogi Co. Ltd. (Osaka) for the generous gift of furanomycin and to Dr. Y. Arata of University of Tokyo for his valuable discussion. We are also grateful to C. Matsumoto for preparation of E. coli tRNA”“.

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T Kohno, D Kohda, M Haruki, S Yokoyama and T Miyazawaprotein.

charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is

1990, 265:6931-6935.J. Biol. Chem. 

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