Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase
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- Simkhada, D., Oh, T., Kim, E.M. et al. Biotechnol Lett (2009) 31: 147. doi:10.1007/s10529-008-9844-9
The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-d-glucose and UDP-d-glucose, respectively, in negative mode.
KeywordsCalicheamicinDeoxysugarMicromonospora echinospora sp. calichensisThymidylyltransferaseUridylyltransferase
The nucleotide-sugar NDP-d-glucose is a key metabolite in prokaryotes where it serves as a precursor to a large number of modified sugars, such as L-rhamnose (6-deoxyhexose), 6-deoxy-l-talose, 2,6-dideoxyhexose and other deoxyhexoses and deoxypentose (Simone et al. 2001). Numerous studies have demonstrated that the early enzymatic step common to the biosynthesis of all deoxysugars found in antibiotics is the formation of NDP-glucose from NTP and α-d-glucose-1-phosphate from glucose-1-phosphate nucleotidylyltransferase (NDP-glucose synthase). The NDP-glucose synthase is found within gene clusters that encode the biosynthetic enzymes for natural products containing deoxyhexose moieties (Pissowotzki et al. 1991; Merson-Davies and Cundliffe 1994; Marolda and Valvano 1995; Sohng et al. 2002).
The calicheamicin gene cluster contains a single nucleotidyltransferase gene (calS7) for the synthesis of both TDP-sugars (3-methoxy-l-rhamnose; TDP-4-hydroxylamine-2,4,6-trideoxy-d-glucose; TDP-4-thio-2,4,6-trideoxy-d-glucose) and the UDP-sugar (UDP-4-amino-3-O-methoxy-2,4,5-trideoxypentose) (Ahlert et al. 2002). Therefore, the calS7 gene present in the calicheamicin gene cluster is proposed to be involved in the transfer of a nucleotidyl group in glucose-1-phosphate during the biosynthesis of sugar moieties (Fig. 1).
Since CalS7 showed putative thymidylyltransfrase based on BLAST analysis from NCBI database, we individually annotated CalS7 with several proteins which were already characterized as uridyltransferases (Aragäo et al. 2006; Thoden and Holden 2007a, b) in order to know the possible putative products. From sequence analysis it is revealed that CalS7 also showed significance similarity with uridyltransferase from several strains like Corynebacterium glutamicum ATCC 13032 (GenBank accession no. NP_599583), Yersinia pestis CO92 (GenBank accession no. NP_405125) and E. coli K12 (GenBank accession no. NP_415752). Sequence analysis of CalS7 provided us strong bootstrap support for heterologous expression. In order to verify the function of it, we cloned and heterologously expressed in E. coli BL21(DE3) and in vitro enzyme assay was carried out. Our results showed that CalS7 is a key enzyme for the biosynthesis of TDP and UDP-sugar moieties present in calicheamicin.
Materials and methods
Bacterial strains, growth conditions, and plasmids
Escherichia coli XL1-Blue MRF′ was used as a host cell for the preparation of recombinant plasmids, and E. coli BL21(DE3) (Stratagene, USA) was used for the expression of the enzyme. Synthetic oligonucleotides were obtained from the Bioneer Co. (Daejeon, Korea). All chemicals used in this study were purchased from Sigma (St. Louis, USA). The disodium salt of glucose-1-phosphate and the disodium salt of ATP, TMP, UMP, TTP, UTP, CTP, GTP and MgCl2·6H2O were used for the enzyme reaction. The pGEM-T Easy (Promega, USA) and pET32a(+) (Novagen, Germany) vectors were used as vectors for cloning and expression, respectively. For the selection and maintenance of plasmids, E. coli was grown at 37°C in Luria-Bertani (LB) broth or on an agar plate supplemented with the appropriate amount of antibiotics when necessary (ampicillin up to 50 μg ml−1). The pET15b plasmid containing the pBR322 origin of replication and the ampicillin resistance gene was used to clone the TMP kinase, and the pET24ma vector containing the p15A origin and kanamycin resistance gene was used to clone acetate kinase (E.C.184.108.40.206). TMP kinase (E.C.220.127.116.11) and acetate kinase were amplified by polymerase chain reaction (PCR) using the genomic DNA of E. coli K12 as a template. All of the genes were cloned under the control of the T7 promoter (Oh et al. 2003).
DNA manipulation and construction of pCalS7
DNA manipulations, restriction endonuclease digestion, and ligation were carried out according to standard protocols. Lysozyme treatment of M. echinospora and phenol-chloroform extraction were performed as described by Kieser et al. (2000). The recombinants were constructed for E. coli. Two synthesized oligonucleotide primers, CalS7F (5′-GCT GAATTC ATG CGT GGT GTT TTG CT-3′) and CalSR (5′-ATT AAGCTT TCA CCC GAC GGC GGC C- 3′), were used to amplify calS7. The PCR product (894 bp) was cloned into the EcoRI and HindIII sites of pET32a(+) in order to generate pCalS7. To verify that no mutation had been introduced during the PCR amplification, the PCR products were cloned into the pGEM-T Easy vector and sequenced before cloning into the expression vector. PCR was performed using a thermocylcer (Takara, Japan) under the following conditions: 30 cycles of 30 s at 94°C, 1 min at 60°C, and 1 min at 72°C. The recombinant expression vector was introduced into E. coli BL21 (DE3) by heat-pulse transformation, and the antibiotic resistant transformants were selected.
Expression and purification of CalS7
Escherichia coli BL21 (DE3) harboring the recombinant plasmid pCalS7 was grown in 3 ml LB culture medium. The overnight culture medium was transferred to 50 ml fresh LB medium containing ampicillin and then grown at 37°C and 250 rpm. At an OD600 of 0.6, IPTG was added to give 0.4 mM, and the incubation was continued for 20 h at 20°C. Thawed cell pellets were harvested by centrifugation at 6,000 × g for 10 min. They were also washed twice with 20 ml of 50 mM Tris/HCl (pH 7.5) and sonication buffer containing 1 mM PMSF, 1 mM DTT, 100 mM EDTA (pH 8), 1 M MgCl2, 50 mM Tris/HCl (pH 7.5) and 10% glycerol. The cells were lysed by sonication on ice with an ultra-sonicator. The debris was removed by centrifugation at 12,000 × g for 40 min at 4°C.
The His-tagged CalS7 fusion soluble protein was purified by Co2+ affinity chromatography (TALON Purification Kit, Clontech, USA) according to the instructions supplied by the manufacturer. The purified fractions were desalted by dialysis and concentrated using Centricon (Ultracel YM-10, Millipore). The molecular weight of the proteins was analyzed and determined by 12% (v/v) SDS-PAGE using standard molecular weight protein markers. The separating and stacking gels consisted of 12 and 5% polyacrylamide, respectively. Protein concentration was determined according to the Bradford method using the Bio-rad protein assay with bovine serum albumin (BSA) as the standard, and a protein concentration of 65 μg/ml was obtained.
Enzyme assay and analysis of CalS7
A typical enzyme assay condition for CalS7 involves 100 μl containing 50 mM Tris/HCl (pH 7.5), 5 mM NTP, 5 mM glucose 1-phosphate, 1.8 U inorganic pyrophosphase, and 25 mM MgCl2. The reaction was carried out for 30 min at 37°C. The reaction was quenched by heating the sample at 90°C for 45 s, followed by centrifugation for 10 min at 4°C. The supernatant was then obtained for HPLC analysis. The concentrations of UMP/TMP, acetyl phosphate, glucose 1-phosphate, ATP and MgCl2 used for the one-pot enzymatic assay of CalS7 were 5, 50, 45, 1 and 20 mM, respectively. The reaction was initiated by using 0.5 U TMP kinase, 50 U acetate kinase and glucose-1-phosphate nucleotidylyltransferase (CalS7) at 37°C for 90 min (Oh et al. 2003). The reaction was stopped by heating the reaction mixture at 90°C for 45 s, and the precipitate was removed by centrifugation at 12,000 × g for 10 min. The supernatant was analyzed by electrospray ionization-mass spectroscopy (ESI/MS) (Thermo Finnigan, USA) and HPLC. HPLC analysis was carried out at 254 nm using a C18 column (C18 XTerra RP-18, 5 μm 5 × 250 mm, Waters). Isocratic elution was with 100 mM potassium phosphate buffer (pH 7.0) containing 8 mM tetra-n-butyl ammonium hydrogen sulfate and methanol (95:5 v/v) at 1 ml min−1.
Results and discussion
The PCR product (894 bp) of calS7 was cloned into the EcoRI/HindIII sites of pET32a(+) to generate pCalS7. The recombinant pCalS7 was transferred and expressed in a heterologous host strain, E. coli BL21(DE3). Expression with His-fusion protein in soluble form was achieved at 20°C after induction with 0.4 mM IPTG for 20 h. During the purification process, the target proteins were completely eluted at a concentration of 100 mM imidazole in potassium phosphate buffer (pH 6.0), and the molecular weights of the denatured proteins determined by SDS-PAGE analysis (Fig. 2c) were in good agreement with those of the calculated values of ~46 kDa.
Conversion ratio of several nucleotides to their respective sugar
Nucleotides (5 mM)
Relative activity with glucose 1-phosphate (5 mM)
1 ± 0.02
1.08 ± 0.03
0.008 ± 0.00
0.001 ± 0.00
0.32 ± 0.01
In addition, a one-pot enzymatic assay was also carried out using TMK, ACK and CalS7, starting with UMP and TMP to obtain UDP-glucose and TDP-glucose, respectively. TMP is the starter substrate for TDP-glucose, whereas UMP is the starter substrate for UDP-glucose. In the presence of thymidylyl kinase, TMP/UMP is converted into TDP/UDP, which provides further direction for the formation TTP/UTP and leads to the production of the final products in the presence of CalS7 (Fig. 2d). Acetate kinase was used in excess to ensure the efficient regeneration of ATP and to prevent the accumulation of UDP and TDP. The enzyme assay was carried out for various time periods in order to determine the conversion rate. The reaction was stopped by heating at 90°C for 45 s and the supernatant was analyzed by HPLC.
The calicheamicin gene cluster contains a single nucleotidyltransferase gene (CalS7) used for the synthesis of both TDP- and UDP-sugars. Gene analysis revealed that the conserved domains that are responsible for thymidylyltransferase and uridylyltransferase are present. Nucleotide reactions and one-pot synthesis were also carried out for the characterization of CalS7 by HPLC and ESI/MS analysis. The results showed that CalS7 is the nucleotidyltransferase gene involved in the biosynthesis of deoxyhexose and deoxypentose sugars in calicheamicin. This research provides the precursor for the biosynthesis of 3-methoxy-l-rhamnose containing TDP-4-hydroxylamine-2,4,6-trideoxy-d-glucose, TDP-4-thio-2,4,6-trideoxy-d-glucose, and UDP-4-amino-3-O-methoxy-2,4,5-trideoxy pentose, and it also advances the production of other activated nucleotide-linked sugars, which are absolutely required for the biosynthesis of the unusual deoxysugars in certain antibiotics.
This study was supported by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0720260) and Korean Research Foundation (KRF) by providing research scholarship for a Ph D scholar.