Study on the putative active site of Enterococcus faecalis prolyl-tRNA synthetase editing domain by methods of site-directed mutagenesis

The maintenance of amino acid specificity by aminoacyl-tRNA synthetases can require the hydrolysis of missynthesized products that is known as amino acid editing. Bacterial prolyl-tRNA synthetase includes a special editing domain, that deacylates alanyl-tRNA, and so exhibits post-transfer editing activity. The mechanism of tRNA-dependent editing by prolyl-tRNA synthetase has to be defined. The present work aim is to study the structure of the active site of enterobacteria E. faecalis prolyl-tRNA synthetase editing domain. The amino acids positions E218, T257, K279, G331, S332, G334, and H366 have been chosen for the site-directed mutagenesis (alanine scanning). An editing activity of the mutants was compared with the wild type prolyl-tRNA synthetase. Three amino acid residues, important for the editing activity, K279, G331 and H366, were revealed. This data are consistent with the existing suppositions about the structure of bacterial prolyl-tRNA synthetase deacylating active site.

In tro duc tion.The main te nance of amino acid spec ific ity by aminoacyl-tRNA syn the tas es may re quire not only spe cific rec og ni tion of amino acid, but also hy droly sis of missynthesized prod ucts that is known as amino acid ed it ing.There are two ways of ed it ing, namely, hy dro ly sis of missynthesized aminoacyl-adenylate (pre-trans fer ed it ing) and hy droly sis of missynthesized aminoacyl-tRNA (post-transfer editing) [1].
Ac tu ally, the mech a nisms of post-trans fer ed it ing by aminoacyl-tRNA syn the tas es of the first struc tural class are rather well-known [2][3][4], while the stud ies on anal o gous mech a nisms for aminoacyl-tRNA syn thetas es of the sec ond struc tural class, i.e. phenylalanyl- [5] and treonyl-tRNA syn the tas es [6], are still in progress.How ever, the prolyl-tRNA syn the tas es post-trans fer ed it ing has been poorly investigated.
Bac te rial prolyl-tRNA syn the tas es are able to perform the pre-and post-trans fer alanine ed it ing [7], which takes place in spe cial ised ed it ing do main, called INS (in ser tion) [8,9].The cur rent re sults of site-directed mu ta gen e sis (for the Esch e richia coli en zyme) [10] and struc tural data (for the E. faecalis en zyme) [11] al lowed us to make some as sump tions about lo cation and struc tural or gani sa tion of the bac te rial prolyl-tRNA synthetase deacylating site.For the E. coli en zyme, a par tic u lar im por tance for the ef fi ciency and spec i fic ity of post-trans fer ed it ing of con ser va tive lysine K279 and con ser va tive histidine H369 has been shown and their par tic i pa tion in the for ma tion of deacylating ac tive site has been sup posed [10].Based on the struc tural data a pos si ble role of con ser va tive lysine K279, glycine G331, and histidine H366 as struc tural and func tional el e ments of the E. feacalis deacylating ac tive site has been sug gested [11].The aim of cur rent work was to ver ify these sug ges tions as well as to com pare the de gree of sim i lar ity be tween the post-trans fer ed it ing mech a nisms for the en zymes of phylo gen eti cally dis tant bac te ria: E. faecalis (Firmicutes type) and E. coli (Proteobacteria type).
Ma te ri als and Meth ods.The mu ta gen e sis kit (Stratagene,USA), plasmid DNA ex trac tion kit (Qiagene, USA), chro mato graphic ma trixes (Pharmacia Biotech, Swe den; Toyo Soda, Ja pan), amino ac ids (Pierce, France), ra dio ac tively marked sub stances (Amersham, UK), fibre glass fil ters (Whatman, USA), PEI-cel lu lose (Merck, Ger many) were used in the work.Cre ation of chi me ric tRNA ProAla and mu tant forms of E. faecalis prolyl-tRNA synthetase.Mu ta gen e sis of E. feacalis tRNA Pro gene in serted into pUC18 vec tor, con tain ing T7-pro moter, in or der to in tro duce the recog ni tion el e ments of alanyl-tRNA synthetase into its se quence (Fig. 1), was per formed by QuickChange method (Stratagene) [12] us ing poly mer ase chain re action (PCR).All mu tant genes were checked by sequenc ing.In vi tro transription and iso la tion of tRNA ProAla mu tants were per formed sim i larly to those for E. feacalis tRNA Pro as pre vi ously de scribed in [12], ex cept for the co-ex pres sion of tRNA with cys-hydrolytic ribosyme.
Mu ta gen e sis of E. faecalis prolyl-tRNA synthetase gene was ac com plished by QuickChange method (Stratagene) [13], iso la tion and pu ri fi ca tion of mu tant pro teins were done as de scribed in [12].
Anal y sis of aminoacylation.130 µl of re ac tion mixture con tained 100 mM tris-HCl, pH 8.0, 20 mM MgCl 2 , 0.5 mg/ml BSA, 3 mM ATP, 3 mM proline, 20 µM 14 C-la belled proline (85.0 mCi/mmol), 5 or 10 µM tRNA Pro (CGG) Rhodopseudomonas palustris and 5 nM prolyl-tRNA synthetase or its mu tant forms.Aliquots of 20 µl were taken from the re ac tion mix ture in cu bated at 37°C and put into 200 µl of cold 10% TCA for tRNA and aminoacyl-tRNA pre cip i ta tion.Then pre cip i tates were trans ferred onto fibre glass fil ters, washed with 50 ml 5% TCA, dried and ra dio ac tiv ity counted in liq uid scintillation counter.
Aminoacylation of tRNA ProAla by 14 C-la belled alanine.0.5 ml alanine-tRNA ProAla were ob tained at the fol low ing con cen tra tions of re ac tion mix ture con stit uents: 600 nM alanyl-tRNA synthetase from Thermus thermophilus, 15 µM tRNA ProAla , 0.03 mM alanine, 78 µM 14 C-la belled alanine, 100 mM tris-HCl, pH 7.5, 15 mM MgCl 2 , 0.5 mg/ml BSA, 3 mM ATP.The mix ture was in cu bated at 37°C for 20 min and acid i fied by sodium-ac e tate buffer with sub se quent treat ment by phenol and chlo ro form.Af ter eth a nol pre cip i ta tion the pellet was dried and dis solved in 40 µl of 0.1 M sodium-ac e tate pH 4.0 buffer solution.Alanyl-tRNA ProAla hy dro ly sis as say.60 µl of re ac tion mix ture con tained 60 nM prolyl-tRNA synthetase of E. feacalis or its mu tant forms, 100 mM HEPES, pH 7.0, 10 mM MgCl 2 , 0.1 mg/ml BSA, 2 mM dithiothreitol (DTT), 3 µl of 14 C-alanyl-tRNA ProAla so lu tion, pre pared as de scribed above.Re ac tion was con ducted at 37°C, 5 µl aliquots were with drawn at zero time-point and af ter 1, 2, 3, 5, 10 min of in cu ba tion, put on fibre glass fil ters, sat u rated by 10% TCA.Then fil ters were washed in 5% TCA, dried and ana lysed in liq uid scin til la tion coun ter.
Anal y sis of ATP hy dro ly sis.18 µl of re ac tion mixture con tained 100 mM HEPES, pH 7.5, 25 mM KCl, 10 mM MgCl 2 , 2 mM DTT, 1 mM ATP, 75 µM 14 C-labelled ATP (57.9 mCi/mmol), 500 mM alanine or 250 mM proline, 15 µM tRNA Pro and 2 µM prolyl-tRNA synthetase.Dur ing in cu ba tion at 37°C, 2 µl aliquots were taken out and put on PEI-cel lu lose.Then ATP, ADP, and AMP were sep a rated by method of thin-layer chro ma tog ra phy in 0.75 M po tas sium-phos phate buffer, pH 3.5.Ra dio ac tiv ity of ATP and AMP zones was ana lysed in liq uid scintillation counter.
Re sults and Dis cus sion.The fol low ing po si tions were se lected for alanine scan ning: T257, K279, H366, ho mol o gous to those which were al ready de fined to be im por tant for the post-trans fer ed it ing ac tiv ity of E. coli prolyl-tRNA synthetase [10]; po si tions G331, S332 , sug gested as es sen tial for the ed it ing do main on the ba sis of struc tural data and com puter sim u la tion [11]; po si tions G334 and E218, lo cated in the area of con tact be tween ed it ing do main and syn thetic do main.
To check the ed it ing ac tiv ity of dif fer ent mu tant forms of prolyl-tRNA synthetase in alanyl-tRNA hy dro ly sis we cre ated a hy brid tRNA, rec og nized by both proline and alanine aminoacyl-tRNA syn the tas es.There fore, we in tro duced the rec og ni tion el e ments of tRNA specific for alanine into the sequence of tRNA specific for proline (Fig. 1).
The ob tained chi me ri cal tRNA was aminoacylated by la belled alanine.The ed it ing ac tiv ity of prolyl-tRNA synthetase mu tant forms was es ti mated by the rate of la belled alanyl-tRNA hy dro ly sis.The data of ini tial rate of deacylation re vealed that three out of seven iso lated mu tant forms of the en zyme (K279A, G331A, and H266A) showed sig nif i cant (from 4 to 50-fold) de creas ing of post-trans fer ed it ing ac tiv ity.K279A mu tant dem on strated 2% ac tiv ity com par ing to that of the wild type en zyme, G331A and H366A mu tants -16% and 24% respectively (Fig. 2).
For fur ther in ves ti ga tion of mu tant forms, show ing sig nif i cant de creas ing of the post-trans fer ed it ing ac tivity in deacylation as say, anal y sis of ATP hy dro ly sis, re flect ing over all pre-and post-trans fer ed it ing (Fig. 3), and aminoacylation by ho mol o gous amino acid were con ducted (Fig. 4).The in ves ti ga tion of ATP hy dro lysis in the pres ence of ed ited amino acid (alanine) revealed the same ten den cies to a de cline in the ed it ing ac tiv ity for dif fer ent mu tant forms of the en zyme.At the same time, the de creas ing of aminoacylation ac tiv ity of mu tant forms does not cor re late with the de creas ing of ed it ing ac tiv ity, and is prob a bly caused by in duced conformational changes in the syn thetic do main of prolyl-tRNA synthetase.These re sults al low us to conclude that mu ta tions K279A, G331A, H366A spe cif ically affect the post-transfer editing.
The data ob tained con firm our pre vi ous as sump tion [11] about the struc ture of E. faecalis prolyl-tRNA synthetase deacylating ac tive site, but they do not prove the role of amino acid res i dues T257 and S332, lo cated close to the deacylating ac tive site.K279 and H366, amino acid res i dues of E. feacalis prolyl-tRNA synthetase, which are ho mol o gous to K279 and H369 of E. coli, have sim i lar [10] im por tance for hy dro ly sis of alanyl-tRNA ProAla which em pha size the sim i lar ity of post-trans fer ed it ing mech a nisms for prolyl-tRNA synthe tas es of both bac te ria, in spite of their be ing somewhat phylogenetically distant.Struc tural data show that a side chain of lysine K279 is di rected out side from all other amino ac ids of pu ta tive ac tive cen tre and prob a bly par tic i pates in the bind ing of ac cep tor end of alanyl-tRNA Pro [11].Sim ilarly to E. feacalis prolyl-tRNA synthetase, a sub sti tution of lysine K279 to alanine in the en zyme of E. coli [10] has the high est im pact on the post-trans fer ed it ing ac tiv ity com pared to all stud ied amino acid res i dues, Ap par ently, this res i due is po si tion ing the sub strate in the ac tive cen tre of the ed it ing do main, there fore, playing cru cial role in the post-transfer editing by enzyme.
Histidine H366 as well as glycine G331, form ing hy dro gen bond with its side chain, per haps, main tains an op ti mal struc ture of the deacylating ac tive cen tre.
The sub sti tu tion of histidine H369 to alanine or cysteine in E. coli en zyme re sulted not only in dra matic de crease in the post-trans fer ed it ing ac tiv ity, but also in dis rup tion of deacylation spec i fic ity and hy dro ly sis of prolyl-tRNA Pro as well [10].The rec og ni tion of a big ger sub strate by such mu tant forms of the en zyme may point out on the dis rup tion of the pocket in teg rity, where the ed it ing amino acid res i due is lo cated [10,11].The same event may in flu ence both sub strate binding and ca tal y sis ef fi ciency which ex plains the decreasing in the post-transfer activity.
In con clu sion, we can sug gest that it is un likely for a side chain of any stud ied amino acid res i due to par tici pate di rectly in the ca tal y sis of deacylation re ac tion.At the same time one can not deny that chem i cal groups of the main chain some of these amino ac ids play a signif i cant role in co or di na tion of the ion or wa ter mol ecules, that are re spon si ble for ca tal y sis.In any case, the mech a nism of alanyl-tRNA Pro deacylation in the ed iting do main ac tive cen tre of bac te rial prolyl-tRNA synthetases is yet to be defined.Ê. Ñ. Áî ÿð øèí, ².À. Êðèê ëè âèé, Î. Â. Ðàºâñüêèé, À. Î. Õ³ì³í, Ã. Ä. ßðåì ÷óê, Ì. À.Òó êà ëî Ïå ðåä áà ÷ó âà íèé àê òèâ íèé öåíòð ðå äà ãó þ ÷î ãî äî ìå íó ïðîë³ë-òÐÍÊ ñèí òå òà çè áàê òåð³¿ Enterococcus faecalis

Fig. 1 .
Fig.1.Mutant E. faecalis tRNA Pro (tRNA ProAla ).Recognition elements for alanyl-tRNA synthetase introduced in the sequence of wild type tRNA Pro are indicated.Nucleotides of anticodon are underlined.

Fig. 2 .
Fig.2.Post-transfer editing activity of wild type and mutant forms of E. faecalis prolyl-tRNA synthetase.Initial rate of deacylation reaction catalyzed by wild-type enzyme was taken as 100%.