Patent Application
20080064646
Reference will be made to the legends of the figures below in the remainder of the description.
a) the “de novo” synthesis of DNA from simple precursors.
The abbreviations used are as follows:
b) the conservation or recycling route which is much less costly in terms of energy and involving transfer reactions of sugar from preformed bases (originating from the hydrolytic degradation of amino acids and nucleotides). The abbreviations used are as follows (enzymes represented by their corresponding genes):
Lactobacillus fermentum, Lm: leuconostoc mesenteroides, Pro mar: prochlorococcus marinus
The synthesis of the nucleosides in E. coli can be carried out according to two processes; [Agnete MUNCH-PETERSEN (1983). “Metabolism of nucleotides, nucleosides and nucleobases in microorganisms” published by Academic Press] (see
Two classes of enzymes exist which catalyze the transfer of a 2-deoxyribosyl to a nitrogenous base; see hereafter and [Jane R. HANRAHAN & David W. HUTCHINSON (1992). “The enzymatic synthesis of antiviral agents”. Journal of Biotechnology; vol. 23; 193-210. The latter are sometimes used for the synthesis of nucleoside analogues].
The N-deoxyribosyl transferases catalyze the cleavage of the glycosidic bonds of the 2-deoxynucleotides. They are present in certain micro-organisms which possess little or no purine and pyrimidine phosphorylase (lactobacilla for example) [6-8]. They participate in the recycling of nucleotides.
Reactions catalyzed according to the type of enzymes
Two types of enzyme have been characterized, [José HOLGUIN & Robert CARDINAUD (1975). “Trans-N-Deoxyribosylase: substrate-specific studies”. European Journal of Biochemistry; vol. 54; 515-520].
Purine deoxyribosyl transferase or NTD1:
It catalyzes exclusively the reversible transfer of a sugar from a puric base (donor base) to another purine base (acceptor base).
(1): (d)R-pur1+pur2 pur1+(d)R-pur2
Pyrimidine/Purine deoxyribosyl transferase or NTD II
It mostly catalyzes the transfer between purine and pyrimidine according to the following reversible equations:
Reaction Mechanism (
If we keep to what is known from Lactobacillus delbruckii, NTD II would react according to a “ping-pong-bi-bi” mechanism which would involve two substrates and two products [Jose HOLGUIN & Robert CARDINAUD (1975). “Trans-N-Deoxyribosylase: Purification by affinity chromatography and characterisation”. European Journal of Biochemistry; vol. 54; 505-514; C. DANZIN & Robert CARDINAUD (1974). “Deoxyribosyl transfer catalysis with trans-N-deoxyribosylase. Kinetic studies of purine to purine trans-N-deoxyribosylase. European Journal of Biochemistry; vol. 48; 255-252; C. DANZIN & Robert CARDINAUD (1976). “Deoxyribosyl transfer catalysis with trans-N-deoxyribosylase. Kinetic studies of purine (pyrimidine) to purine (pyrimidine) trans-N-deoxyribosylase. European Journal of Biochemistry; vol.62; 356-372].
It is assumed that the sugar of the donor nucleoside (dBase1) binds to the enzyme covalently. An intramolecular reaction within this binary complex allows the cleavage of the β-glycosidic bond and the formation of a ternary complex E-deoxyribosyl-Base1 followed by the release of the first product (Base1). The acceptor base (Base2) then binds to the binary intermediate and after intramolecular reaction on the active site of the enzyme, the second product (dBase2) is released. The enzyme can then carry out another catalysis.
Physico-chemical Properties
In Lactobacillus delbruckii, the two enzymes have a similar molecular weight (evaluated at about 100 kDa) but they differ in their thermal stability (activity observed up to 45° C. for NTD I et 55° C. for NTD II) and their optimum pH (6.4 for NTD I and 6.0 for NTD II).
The ntd gene of Lactobacillus delbruckii coding for NTD II with a length of 471 bp codes for the synthesis of a protein with 157 amino acids and total molecular weight of 110 kDa [William J. COOK, Steven A. SHORT & Steven E. EALICK (1990). “Crystallization & preliminary X-ray investigation of recombinant Lactobacillus leichmanii nucleoside 2-deoxyribosyl transferase”. The Journal of Biological Chemistry; vol. 265; No. 5; 2682-2683]. The crystalline structure of the enzyme NTD II of L. delbruckii was determined with a resolution of 2.5 Å. This is a hexamer (trimer of dimers) constituted by six identical sub-units of 18 kDa. Each sub-unit possesses in the centre a parallel β-sheet comprising five strands of various lengths and surrounded by four a helices arranged asymmetrically. Each comprises an active site, but the six catalytic centres, approximately 20 Å distant in pairs, require the participation of the side chains of the adjacent sub-units [Shelly R. ARMSTRONG, William J. COOK, Steven A. SHORT & Steven E. EALICK (1996). “Crystal structures of nucleoside 2-deoxyribosyl transferase in native & ligand-bound forms reveal architecture of the active site”. Structure; vol. 4; No. 1; 97-107]. The latter are involved in the positioning of the catalytic amino acid (glutamate 98) [David J. T. PORTER, Barbara M. MERRIL & Steven A. SHORT (1995). “Identification of the active site nucleophile nucleoside 2-deoxyribosyl transferase as glutamic acid 98”. The Journal of Biological chemistry; vol. 270; No. 26; 15551-15556].
Enzymatic Synthesis of Nucleoside Analogues
The highly stereospecific transfer reactions, in the presence of an NTD I or NTD II transferase, exclusively produce the β anomer of the nucleoside (which avoids the stage of separation of the a and β isomers).
The enzyme possesses a great specificity vis-à-vis 2′-deoxyribonucleotides but tolerates a large number of modified analogues on the sugar or on the base. Thymidine and cytosine seem to be the most effective donors of sugar. On the other hand the transfer can be made to a large panel of acceptor bases. There can for example be mentioned the purines substituted in position 6 [D. BETBEDER, D. W. HUTCHINSON & A. O. RICHARDS (1989). “The stereoselective enzymatic synthesis of 9-β-D-2′,3′-dideoxynucleosides of N(6)substituted purines”. Antiviral Chem. Chemother; vol.17; 4217-4222] and dYTP.
dYTP:
The imidazole-4-carboxamide called Y was proposed as simplified purine. This analogue has the formula:
It has been reported that the nucleotide dYTP could be substituted for dATP or dGTP during the copying of the DNA which introduces mutations. There can also be mentioned the compounds described in WO 01/96354 (Institut Pasteur) of general formula:
The NTD enzymes prove to be capable of marginally catalyzing the exchange reaction between a 2′,3′-dideoxyribose and an acceptor base: dd-1′-Base1+Base2Base1+dd-1′-Base2 (dd=2′,3′-dideoxyribose)
Nevertheless the speed of this transfer remains very low compared to that characterizing the exchange of deoxyriboses.
The 2′,3′-dideoxyribonucleotides are evidently useful as chain terminators in the sequencing procedures. Furthermore, 2′,3′-dideoxyadenosine (ddA) and 2′,3′-dideoxyinosine (ddl) are used for therapeutic purposes in particular in the case of the AIDS virus: these analogues effectively inhibit the replication of HIV (human immunodeficiency virus) [H. MITSUYA & S. BRODER (1987).“Strategies for antiviral therapy in AIDS”. Nature; vol. 325; 773-778].
To this end, the invention provides a novel method for obtaining mutants of the NTD II enzyme in order to select mutant enzymes of L. fermentum which have a stronger specificity towards the 2′,3′-dideoxyribonucleosides than the native enzyme.
The PAK9 E. coli strains are cultured in Luria-Bertani (LB) medium or in minimum MS medium (Richaud et al. 1993). The antibiotics kanamycin, Km and chloramphenicol Cm, are used at a final concentration of 25 μg/ml; tetracycline, Tc and gentamycin, Gm, 10 μg/ml. The nucleosides and bases are used in the culture media at a final concentration of 0.3 mM. The molecular biology techniques are carried out according to Sambrook et al. (1989)
The amplification products are purified using QIAquick PCR purification (QIAgen)
The DNA fragments purified on agarose gel are extracted using the Jetsorb Kit (Genomed) or the QIAquick gel extraction kit (QIAgen). The plasmidic DNA is purified using the QIAprep spin miniprep kit (QIAgen)
The strain PAK9 (MG1655 ΔpyrC::Gm, AcodA::Km, cdd::Tn10) is available from the CNCM (Collection Nationale de Culture des Microorganismes) at the Institut Pasteur, 25-28 rue du Dr Roux 75224 Paris cedex 15, under No.1-2902.
The vector pSU19N was obtained by site-directed mutagenesis of the plasmid pSU19 [B. BARTOLOME, J. JUBETE, E. MARTINEZ & F. DE LA CRUZ (1991) “Constructions and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives” Gene; vol. 102; 75-78; E. MARTINEZ, B. BARTOLOME & F. DE LA CRUZ (1988) “pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids” Gene; vol 68(1); 159-b 162] using the oligonucleotides
An ntd gene of L. fermentum was amplified by PCR from the plasmid pLF6 used here as DNA matrix. The plasmid pLF6 propagated from the E. coli strain PAK6 deposited at the CNCM on 2 May 2001 under the reference 1-2664, contains a fragment Alu I of 1.36 kb of the gene encoding the N-deoxyribosyl transferase of type 11 originating from the strain L. fermentum CIP102780T. In order to amplify this DNA fragment, the following oligonucleotides were used:
then the amplified fragments were digested by the restriction enzymes BamHI and Ndel and cloned in the vector pSU19N. In this construction, the expression of the protein is under the control of the lac promoter.
The primers T7prom (5′-TTAATACGACTCACTATAGGGG)(SEQ ID No.9) and T7term (5′-GCTAGTTATTGCTCAGCGG) (SEQ ID No.10) were used to amplify the ntd gene cloned in the plasmid pET24a (Novagen) according to standard amplification conditions using the GeneMorph PCR Mutagenesis Kit (Stratagene, USA). The amplification parameters: 1 cycle of 5′ at 95° C., 30 cycles each comprising the following three stages: 30″ at 95° C., 30″ at 51.5° C., 1′ at 72° C., then a cycle of 10′ at 72° C. The concentrations of DNA matrix used: 10 ng and 10 pg.
The purified amplification products are digested for 2 hours at 37° C. by the restriction enzymes BamHI and Ndel. After migration at 150V for 45 minutes, they are purified by 1% agarose gel extraction using the QIAquick gel extraction kit (QIAgen).
The plasmid pSU19N is digested by the same enzymes and purified according to the same procedure.
The ligations produced in a volume of 20 μl comprise 15 ng of the amplification products, 50 ng of pSU19 digested by BamHI-HindIII, 2 μl of 10× concentrated reaction buffer of T4 DNA ligase and 6U of T4 DNA ligase. The reaction is carried out at 16° C. for 18 hours.
The ligation products are then dialysed on Millipore filter (0.05 μm; 13 mm) for 30 minutes then used to transform the strain PAK9, prepared according to the protocol described by Dower et al. (1987), by electroporation. 1 to 5 μl of ligated DNA mixed with 50 μl of the strain PAK9 in a 2 mm cuvette are subjected to a charge of 2.5 kV. After incubation for one hour at 37° C. in 1 ml of LB medium supplemented with uracil (0.3 mM), two successive washings with 1 ml of 1× MS medium are carried out. 450 μl of suspension are plated on mineral glucose agar medium supplemented with Cm, ddU and C. The dishes are incubated at 37° C. for 4 days. The selected colonies are then isolated on the same medium.
The plasmid DNA of the isolated colonies is prepared from cultures in LB medium supplemented with Cm and U. The sequencing of the plasmids was carried out by the company MWG-BIOTECH.
The sequencing of the plasmids present in the selected transformants of PAK 9 made it possible to identify a mutation in the sequence (ntd) having the effect of substituting a residue T for the residue A in position 15 in the corresponding protein sequence (SEQ ID No. 2) (mutation called A15T).
The precultures are obtained after inoculation of an isolated colony in 5 ml of LB medium containing Cm and U for the strain PAK9 followed by incubation overnight under stirring at 37° C.
The next day, 15 ml of LB medium containing Cm and U are inoculated at an OD600=0.01. The cultures are then incubated at 37° C. up to an OD comprised between 0.8 and 1.
The cells are then centrifuged at 4000 rpm for 30 minutes at 4° C., the pellet is resuspended in 10 ml of phosphate buffer (Na2HP04+NaH2PO4) at 50 mM (pH =7.5). After centrifugation, the pellet is resuspended in 1 ml of the same buffer. The cells, preserved in ice, are then subjected to three cycles of 30 s of sonication and 30 s of rest. After centrifugation at 12000 rpm for 2×15 minutes at 4° C., the supernatants are recovered and stored at −20° C.
50 μl of enzymatic extract is added to 200 μl of 100 mM citrate buffer, pH 6.44, in the presence of 3 mM ddU or dU final and of 1 mM C final for the strain PAK9, the whole is incubated at 37° C. The progress of the reaction is monitored by TLC (silica; eluent: MeOH—CH2Cl2 (20/80)). The products are revealed under UV, and the sugars revealed by Zücker reagent. The disappearance of the substrates and the formation of the products were also quantified by HPLC analysis. The different products are separated by analytical HPLC with a reversed-phase column (100-5C18) using a flow rate of 1 ml/min and a linear gradient of 5-25% CH3CN in a 10 mM triethyl ammonium acetate buffer at pH 7.5 for 20 minutes.
The oligonucleotides:
were used as primer in an amplification reaction under standard conditions using the ntd gene of L. fermentum cloned in pSU19 (pLF6) as DNA matrix. The amplification product was digested by the restriction enzymes Ndel and BamHI for 2 hours at 37° C., purified on agarose gel and inserted into the plasmid pET24a digested by the same enzymes then the ligation mixture is used to transform the strain β 2033. The plasmid DNA from the colonies was prepared and digested by the enzymes Ndel and BamHI. Those, the sequence of which was correct, were used to transform the strain BL21 (DE3)/piysS (Novagen). The plasmid DNA of the mutant pSU19NLFA15T selected previously was prepared then digested by the enzymes Ndel and BamHI. The corresponding fragment Ndel-BamHI was then inserted into the plasmid pET24a digested by the same enzymes in order to produce the expression plasmid pETLFA15T useful to the expression of the mutated protein. A strain of E. coli transformed using the plasmid pETLFA15T was deposited at the CNCM on 22 Mar. 2004 under accession number 1-3192. The overproduction of the two N-deoxyribosyl transferases, native and mutated, was obtained from cultures of this strain in 500 ml of LB medium supplemented with Km and Cm. These cultures were induced at an OD600=0.6 by the addition of IPTG (0.4 mM), the incubation being continued for 2 hours 30 minutes at 37° C.
The cells are then centrifuged for 15′ at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at −20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90′ at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30′ at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.
The measurement of the enzymatic activities is carried out as described in paragraph 4.2.
The transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.
Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur ddR-Pur+Pyr as well as dR-Pyr+Pur
dR-Pur+Pyr.
The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.
S
Lactobacillus gasseri
T
Oenococcus oeni MCW
Leuconostoc mesenteroides ATCC 8293
Lactobacillus plantarum WCFS1
Lactococcus lactis IL 1403
The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CdC+T, ddT+C
ddC+T and d4T+C
d4C+T were evaluated starting from crude extracts or purified proteins.
The results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.
dC + T.
Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.
The selected enzyme is therefore used in the enzymatic synthesis of 2′,3′-dideoxynucleosides and 2′,3′-dideoxy, 2′,3′-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3′-fluoro) 2′,3′-dideoxy, 2′,3′-didehydro-4′-thio-Nucleosides comprising or not comprising radioelements.
As shown by the alignment of
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| Number | Date | Country | Kind |
|---|---|---|---|
| 0403319 | Mar 2004 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR05/00743 | 3/29/2005 | WO | 00 | 9/29/2006 |
