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Staphylococcus aureus TMPK and TK showing distinct structural differences with human TMPK and TK – A probable explanation in the pathogenesis
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 9 e9 5
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Staphylococcus aureus TMPK and TK showing distinct structural differences with human TMPK and TK e A probable explanation in the pathogenesis Hari Prasad Osuru a, Nanda Kumar Yellapu b, Santhosh Kumar Pasupuleti a, Reddy Obulam Vijaya Sarathi c, Abhijit Chaudhary d, Sarma Potukuchi Venkata Gurunadha Krishna e,* a
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati 517502, India c Department of Biochemistry, Sri Venkateswara University, Tirupati 517502, India d Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India e Head, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Alipiri Road, Tirupati 517507, India b
article info
abstract
Article history:
Background: The biosynthesis of dTMP in Staphylococcus aureus is catalyzed by thymidine ki-
Received 9 November 2012
nase (TK) whose concentration plays critical role in the formation of small colony variants.
Accepted 21 January 2013
This dTMP is phosphorylated to dTDP by thymidine monophosphate kinase (TMPK) for
Available online 7 March 2013
subsequent synthesis of dTTP in the bacteria. Owing to the importance of these enzymes in S. aureus TMPK and TK genes were characterized and compared with human TMPK and TK.
Keywords:
Method: The TMPK and TK genes were PCR amplified from the chromosomal DNA of S.
KEN box
aureus ATCC12600 and cloned, sequenced, expressed and characterized. The annotated
P-loop
protein sequence of TMPK and TK were compared with other bacterial and human TMPK
RMSD
and TK. The S. aureus TMPK and TK structures were retrieved from PDB and compared with
TMPK
human TMPK and TK structures.
TK
Results: The gene sequences (FJ415069 and FJ232923) showed complete homology with TMPK and TK genes present in all S. aureus strains. The 3D comparative structural analysis between S. aureus and human TMPK and TK showed very close homology between them as indicated from RMSD values of 0.913 A and 1.336 A respectively. However, presence of G in the P-loop region of S. aureus TMPK whereas R was found in human TMPK similarly, absence of KEN box in S. aureus TK and present in human TK. Conclusion: S. aureus phosphorylates only L enantiomer of dTMP while human TMPK selectively phosphorylates the D enantiomer of dTMP and its analogs probably explains this enzyme of S. aureus specific target for the development of new antimicrobial agents and conspicuous absence of human TK in active cells probably explains the rapid proliferation of S. aureus in human host. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. Tel.: þ91 8772287777x2394,2395; fax: þ91 8772286803. E-mail address: [email protected] (S. Potukuchi Venkata Gurunadha Krishna). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.01.021
90
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j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 9 e9 5
Introduction
Staphylococcus aureus is the leading causative pathogen of hospital-acquired infections, which are increasingly resistant to antibiotics.1e3 Relapse episodes of S. aureus infections have shown to possess reduced colony morphology and are stated as small colony variants (SCV) which are shown having elevated intracellular dTMP levels resulting in increased virulence.4e6 Thymidine kinase (TK) is the key enzyme in the pyrimidine salvage pathway, catalyzes the phosphorylation of thymidineethymidine 50 -monophosphate (TMP).7 TK is important for cells engaged in active DNA synthesis and is regulated by feedback control mechanism mediated by thymidine 50 -triphosphate.8 Thus, formed dTMP is converted to dTDP by thymidine monophosphate kinase an enzyme which is junction between salvage and de novo biosynthesis. Therefore, any variation in de novo or in salvage pathway the TMPK activity is very much influenced. TK and TMPK have been characterized in many bacteria and eukaryotes.9e12 NMP kinases exhibit a protein fold featuring a central fivestranded b-sheet surrounded by helices.13 The protein can be divided into three parts, namely, the CORE region, the NMPbinding region, and the LID region. The CORE region is the most conserved among NMP kinases, comprising mainly bsheets with surrounding a-helices, and contains the P-loop, which is the ATP binding site. The NMP-binding domain is largely helical among all NMP kinases except guanylate monophosphate kinases. The LID region covers part of the phosphate donor site. Substrate-induced conformational changes have been observed in various family members of NMP kinases with large domain movements upon binding of one or both substrates.13,14 Distinct differences have been observed between human TMP kinases and bacterial TMP kinases and among various classes of bacteria.9e12 Moreover, human TK is present actively present only in the G phase of the cell whereas, TK is present in large amounts in S. aureus and they normally by pass the ubiquitin mediated proteolysis15 and therefore help the proliferation of S. aureus in the human host. Therefore, the present study is focused on the characterization of TK and TMPK genes of S. aureus, further its comparison with human TMPK and TK.
2.
Materials and methods
2.1.
Culturing of S. aureus
S. aureus ATCC12600 was grown on modified Baird Parker media16,17 at 37 C. After overnight incubation single black shiny colored with distinct zone colony was picked and cultured in brain heart infusion (BHI) broth at 37 C and this culture was used for the extractions of cytoplasm and chromosomal DNA. The cytosolic fraction was used for the TK and TMPK enzyme assay while chromosomal DNA is used for amplification of TK and TMPK genes.16e18
2.2.
TK and TMPK enzyme assays
The enzyme activities were determined at 30 C using coupled spectrophotometric assay on a Cyber lab spectrophotometer USA. One unit of TK activity is defined as the amount of enzyme catalyzing the production of 1 mmol nucleoside monophosphate per minute whereas one unit of TMPK activity is defined as the amount of enzyme catalyzing the production of 1 mmol nucleoside diphosphate per minute. The kinetic parameters Km and Vmax were evaluated from HaneseWoolf plot ([S] vs [S/V]). Protein concentrations in all steps were determined by Bradford 1976 method.17e19
2.3. Cloning and expression of TMP kinase and TK genes from S. aureus ATCC12600 TMPK and TK genes were PCR amplified from the chromosomal DNA following the primers and conditions mentioned in Table 1.3 Thus, amplified products of TK and TMP kinase were purified with NP-PCR purification kit, Taurus Scientific, USA and were sequenced by dye terminating method at MWG Biotech India Ltd and the sequences were deposited at GenBank www.ncbi.nlm.nih.gov. The cloning of TK and TMPK genes were carried out as described earlier.19 The TK and TMPK genes were expressed using 1 mM IPTG from clones HTK and HTM respectively and pure rTK and rTMPK were obtained from respective clones were analyzed and characterized.17e19
Table 1 e PCR primers used in this study and the product size amplified from the chromosomal DNA isolated from S. aureus ATCC12600. Primers
Gene name
Forward HTKF 50 CATGTATGAAACTTAC30 Reverse HTKR 50 CGCGCGACAACGTG
TK
TMH1 forward 50 CATCTCAGCTTTTATAAC30 TMH2 reverse 50 GATGATAGTTTGATACGTG3’
TMPK
PCR conditions
Size of the PCR product (Kb)
Initial denaturation 94 C for 10 min, and for 40 cycles 94 C for 60 s, 42.1 C for 45 s, 72 C for 60 s and final extension at 72 C for 10 min Initial denaturation 94 C for 10 min, and for 40 cycles contain 94 C for 50 s of denaturation, 42.1 C for 45 s of annealing, 72 C for 60 s of extension and final elongation at 72 C for 10 min
0.55
0.58
91
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Table 2 e Kinetics of thymidine monophosphate kinase. S. No. 1. 2.
Source of TMP kinase
Enzyme activity (mMNADH/ml/min)
Vmax (mMNADH/mg/min)
Km (mM TMP)
Cytosolic fraction of S. aureus ATCC12600 Purified rTMP kinase from clone HTM clone.
0.43 0.02 0.52 0.02
32.3 1.5 31.33 1.5
0.85 0.03 1.33 0.04
Values are the mean SD from three determinations.
Table 3 e Kinetics of thymidine kinase. S. No. 1. 2.
Source of TK
Enzyme activity (mM NADH/ml/min)
Vmax (mM NADH/mg/min)
Km (mM thymidine)
Cytosolic fraction of S. aureus ATCC12600 Purified rTK kinase from clone HTK.
6.1 0.26 5.2 0.2
208 3 206.7 1.5
20.83 1.25 18.67 1.5
Values are the mean SD from three determinations.
Fig. 1 e Cloning of Staphylococcus aureus TMPK and TK genes A. PCR amplification of TMPK gene, lane M molecular size marker obtained from Bangalore Genei Pvt. Ltd, lane 1 and lane 2 PCR amplified products. B. PCR amplification of TK gene lane M molecular size marker obtained from Bangalore Genei Pvt. Ltd, lane 1 and 2 PCR amplified products. C. Schematic representation of pQE 30 plasmid vector. D. SDS-PAGE (10%) under reducing conditions of recombinant TMPK and TK from HTM (1) and HTK (2) clones respectively. (1) lane M molecular weight marker obtained from Bangalore Genei Pvt Ltd. Lane 1 uninduced HTM clone cytosolic fraction, lanes 2 and 3 induced cytosolic fraction of HTM clone with 1 mM IPTG. Lane 4 flow through collected after passing induced lysate through nickel metal chelate chromatographic column. Lane 5 recombinant TMPK eluted from nickel metal chelate column. (2) lane M molecular weight marker obtained from Bangalore Genei Pvt Ltd. Lanes 1 and 2 induced cytosolic fraction of HTK clone with 1 mM IPTG. Lane 3 uninduced HTK clone cytosolic fraction and lane 4 recombinant TK eluted from nickel metal chelate column.
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Fig. 2 e Multiple sequence alignment of TK of S. aureus with other bacteria and Homo sapiens. The highly conserved regions are indicated in mustard color, while partially in violet color. A. Multiple sequence alignment of S. aureus TK with bacteria and Homo sapiens TK sequences B. Sequence alignment of S. aureus and human TK sequences. Yellow and purple color sequences highlight the KEN and sequence in humans and absent in S. aureus.
2.4.
In silico analysis of TK and TMPK
TK and TMPK annotated protein sequences of S. aureus ATCC 12600 were analyzed by using the Internet available free softwares e NCBI BLAST, Bio-edit, Mega 4.1 and Clustal X.20e22 The translated TK and TMPK protein sequences were submitted to BLAST-P for similarity searching to find out its homologs.20 Pairwise and multiple sequence alignment were performed using Clustal X.21 The phylogenetic tree produced by the multiple sequence alignment was analyzed by using MEGA 4.1.22 The protein sequences were scanned against Pfam database to identify the conserved domains and family information of the proteins.23 The TK and TMPK structures of S. aureus were retrieved from (PDB IDs: 3E2I and 4DWJ) and were superimposed with human TK and TMPK (PDB IDs: 1XBT and 2XX3) using MATRAS program.24 The extent of homology between the structures was represented by respective RMSD values.
3.
Results and discussion
The TK and TMPK which are prominent enzymes involved in the formation of dTMP and dTDP respectively play critical role
Fig. 3 e Superimposed structures of S. aureus TK structure (PDB ID: 3E2I) (green) with human TK (PDB ID: 1XBT) (blue) were generated by using MATRAS program.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 9 e9 5
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Fig. 4 e A. Multiple sequence alignment of S. aureus TMPK with bacteria and Homo sapiens TMPK, highly conserved regions are indicated by yellow and violet. B. Sequence alignment of S. aureus and human TMPK sequences showing P-loop and LIDregions.
in the proliferation and pathogenesis of S. aureus in the human host especially in relapsed episodes and in SCV.4e6 TMPK is an enzyme which is in junction between de novo biosynthesis and salvage pathway and therefore, obtains substrates from both the pathways. The enzyme kinetics results of TK and TMPK (Tables 2 and 3) indicated that these enzymes are actively present in this pathogen (Supplementary Figs. 1 and 2). The TMPK and TK genes in the clones HTM and HTK respectively were confirmed by PCR using the primers mentioned in Table 1 and the insert in the clones were sequenced (GenBank accession numbers FJ415069 and FJ232923). The pure recombinant proteins eluted from nickel metal chelate agarose column (Bangalore Genei Pvt Ltd) exhibited single band in SDS-PAGE (10%) with a molecular weights of 21 kDa and 20 kDa respectively17 (Fig. 1). The structural superimposition results of S. aureus TK and TMPK and human TK and TMPK structures24 indicated RMSD
values of 0.913 A and 1.336 A respectively showing close homology between the structures (Figs. 3 and 5). However, TMPK structure of S. aureus exhibited typical characteristics of a class II enzyme, containing a G at position x1 of the P-loop whereas R is present in human TMPK and a series of basic residues (R 141, R 147, R 151 and K 144) in the LID region of S. aureus TMPK. Additionally, significant differences in the TMP site of S. aureus TMPK compared to other bacterial TMPKs and human TMPK have been identified.11,25,26 Also, human TMPK selectively phosphorylates the D enantiomer of dTMP and its analogs26 (Fig. 4A and B). This enantioselectivity of nucleosideactivating enzymes most likely have a strong impact on the efficacy and specificity of new antimicrobial agents. Human TK has very close homology with the TK of S. aureus ATCC12600 however, (Fig. 2A and B) humanTK1 has a unique KEN box in the C terminal region which is the binding site for ubiquitin ligase and thereby degrades HTK via an ubiquitin proteasome pathway,15 which is distinctly absent in the S. aureus TK.
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Fig. 5 e Superimposed structures of S. aureus TMPK structure (PDB ID: 4DWJ) (magenta) with human TMPK (PDB ID: 2XX3) (yellow) were generated by using MATRAS programme.
4.
Conclusion
In the present study TMPK and TK genes of S. aureus ATCC12600 have been cloned, expressed and characterized. The TMPK and TK kinetics clearly indicated that TMPK and TK are highly active enzymes in this pathogen and showed very close structural similarities with human TMPK and TK. However, absence of KEN sequence in S. aureus TK aids in the proliferation of this bacteria and the distinct differences observed in the substrate enantioselectivity of human TMPK conclude that dTMP analogs having L specificity could be strong antimicrobial agents. These unique differences correlated with variations in functions probably explains the rapid proliferation of S. aureus in its human host and which can be very serious and life threatening with the infections caused by multi drug resistant strains of S. aureus.
Conflicts of interest All authors have none to declare.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jopr.2013.01.021.
references
1. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339:520e532. 2. Hiramatsu K. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis. 2001;1(3):147e155.
3. Ohta T, Hirakawa H, Morikawa K, et al. Nucleotide substitutions in Staphylococcus aureus strains, Mu50, Mu3 and N315. DNA Res. 2004;11(1):51e56. 4. Kahl BC, Belling G, Becker P, et al. Thymidine-dependent Staphylococcus aureus small colony variants are associated with extensive alterations in regulator and virulence gene expression profiles. Infect Immun. 2005;73(7):4119e4126. 5. Kahl B, Herrmann M, Schulze A, et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis. 1998;177(4):1023e1029. 6. Zander J, Besier S, Saum SH, et al. Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus. Infect Immun. 2008;76(4):1333e1339. 7. Okazaki R, Kornberg A. Deoxythymidine kinase of Escherichia coli. I. Purification and some properties of the enzyme. J Biol Chem. 1964a;239:269e274. 8. Okazaki R, Kornberg A. Deoxythymidine kinase of Escherichia coli. II. Kinetics and feedback control. J Biol Chem. 1964b;239:275e284. 9. Saito H, Tomioka H. Thymidine kinase of bacteria: activity of the enzyme in actinomycetes and related organisms. J Gen Microbiol. 1984;130(7):1863e1870. 10. Haouz A, Vanheusden V, Munier-Lehmann H, et al. Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. J Biol Chem. 2003;278:4963e4971. 11. Kotaka M, Dhaliwal B, Ren J, et al. Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding. Protein Sci. 2006;15:774e784. 12. Carnot C, Wang L, Topalis D, Eriksson S. Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase. Protein Sci. 2008;17:1486e1493. 13. Yan H, Tsai MD. Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv Enzymol Relat Areas Mol Bio. 1999;73:103e134. 14. Sekulic N, Shuvalova L, Spangenberg O, Konrad M, Lavie A. Structural characterization of the closed conformation of mouse guanylate kinase. J Biol Chem. 2002;277(33):30236e30243. 15. Kauffman MG, Kelly TJ. Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis. Mol Cell Biol. 1991;11(5):2538e2546. 16. Stadhouders J, Hassings F, van Aalsten-van Maren NO. A pour-plate method for the detection and enumeration of coagulase-positive Staphylococcus aureus in the Baird-Parker medium without egg-yolk. Netz Milk Diary J. 1976;30:222e229. 17. Hari Prasad O, Nanda Kumar Y, Reddy OVS, Chaudhary Abhijit, Sarma PVGK. Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus. Protein J. 2012;31:345e352. 18. Blondin C, Serina L, Wiesmu¨ller L, Gilles AM, Ba^rzu O. Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Anal Biochem. 1994;220(1):219e221. 19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248e254. 20. Altschul SF, Madden TL, Scha¨ffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389e3402. 21. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2. Bioinformatics. 2007;23(21):2947e2948. 22. Tamura K, Dudley J, Nei M, Kumar S. MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596e1599.
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23. Finn RD, Mistry J, Tate J, et al. The Pfam protein families database. Nucleic Acids Res. 2010;38(Database issue):D211eD222. 24. Kawabata T. MATRAS a program for protein 3D structure comparison. Nucleic Acids Res. 2003;31(13):367e369. 25. Ostermann N, Segura-Pena D, Meier C, et al. Structures of human thymidylate kinase in complex with prodrugs:
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implications for the structure-based design of novel compounds. Biochemistry. 2003;42(9):2568e2577. 26. Li de la Sierra I, Munier-Lehmann H, Gilles AM, Barzu O, Delarue M. X-ray structure of TMP kinase from Mycobacterium ˚ resolution. J Mol tuberculosis complexed with TMP at 1.95 A Biol. 2001;311(1):87e100.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Staphylococcus aureus TMPK and TK showing distinct structural differences with human TMPK and TK e A probable explanation in the pathogenesis Hari Prasad Osuru a, Nanda Kumar Yellapu b, Santhosh Kumar Pasupuleti a, Reddy Obulam Vijaya Sarathi c, Abhijit Chaudhary d, Sarma Potukuchi Venkata Gurunadha Krishna e,* a
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati 517502, India c Department of Biochemistry, Sri Venkateswara University, Tirupati 517502, India d Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India e Head, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Alipiri Road, Tirupati 517507, India b
article info
abstract
Article history:
Background: The biosynthesis of dTMP in Staphylococcus aureus is catalyzed by thymidine ki-
Received 9 November 2012
nase (TK) whose concentration plays critical role in the formation of small colony variants.
Accepted 21 January 2013
This dTMP is phosphorylated to dTDP by thymidine monophosphate kinase (TMPK) for
Available online 7 March 2013
subsequent synthesis of dTTP in the bacteria. Owing to the importance of these enzymes in S. aureus TMPK and TK genes were characterized and compared with human TMPK and TK.
Keywords:
Method: The TMPK and TK genes were PCR amplified from the chromosomal DNA of S.
KEN box
aureus ATCC12600 and cloned, sequenced, expressed and characterized. The annotated
P-loop
protein sequence of TMPK and TK were compared with other bacterial and human TMPK
RMSD
and TK. The S. aureus TMPK and TK structures were retrieved from PDB and compared with
TMPK
human TMPK and TK structures.
TK
Results: The gene sequences (FJ415069 and FJ232923) showed complete homology with TMPK and TK genes present in all S. aureus strains. The 3D comparative structural analysis between S. aureus and human TMPK and TK showed very close homology between them as indicated from RMSD values of 0.913 A and 1.336 A respectively. However, presence of G in the P-loop region of S. aureus TMPK whereas R was found in human TMPK similarly, absence of KEN box in S. aureus TK and present in human TK. Conclusion: S. aureus phosphorylates only L enantiomer of dTMP while human TMPK selectively phosphorylates the D enantiomer of dTMP and its analogs probably explains this enzyme of S. aureus specific target for the development of new antimicrobial agents and conspicuous absence of human TK in active cells probably explains the rapid proliferation of S. aureus in human host. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. Tel.: þ91 8772287777x2394,2395; fax: þ91 8772286803. E-mail address: [email protected] (S. Potukuchi Venkata Gurunadha Krishna). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.01.021
90
1.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 9 e9 5
Introduction
Staphylococcus aureus is the leading causative pathogen of hospital-acquired infections, which are increasingly resistant to antibiotics.1e3 Relapse episodes of S. aureus infections have shown to possess reduced colony morphology and are stated as small colony variants (SCV) which are shown having elevated intracellular dTMP levels resulting in increased virulence.4e6 Thymidine kinase (TK) is the key enzyme in the pyrimidine salvage pathway, catalyzes the phosphorylation of thymidineethymidine 50 -monophosphate (TMP).7 TK is important for cells engaged in active DNA synthesis and is regulated by feedback control mechanism mediated by thymidine 50 -triphosphate.8 Thus, formed dTMP is converted to dTDP by thymidine monophosphate kinase an enzyme which is junction between salvage and de novo biosynthesis. Therefore, any variation in de novo or in salvage pathway the TMPK activity is very much influenced. TK and TMPK have been characterized in many bacteria and eukaryotes.9e12 NMP kinases exhibit a protein fold featuring a central fivestranded b-sheet surrounded by helices.13 The protein can be divided into three parts, namely, the CORE region, the NMPbinding region, and the LID region. The CORE region is the most conserved among NMP kinases, comprising mainly bsheets with surrounding a-helices, and contains the P-loop, which is the ATP binding site. The NMP-binding domain is largely helical among all NMP kinases except guanylate monophosphate kinases. The LID region covers part of the phosphate donor site. Substrate-induced conformational changes have been observed in various family members of NMP kinases with large domain movements upon binding of one or both substrates.13,14 Distinct differences have been observed between human TMP kinases and bacterial TMP kinases and among various classes of bacteria.9e12 Moreover, human TK is present actively present only in the G phase of the cell whereas, TK is present in large amounts in S. aureus and they normally by pass the ubiquitin mediated proteolysis15 and therefore help the proliferation of S. aureus in the human host. Therefore, the present study is focused on the characterization of TK and TMPK genes of S. aureus, further its comparison with human TMPK and TK.
2.
Materials and methods
2.1.
Culturing of S. aureus
S. aureus ATCC12600 was grown on modified Baird Parker media16,17 at 37 C. After overnight incubation single black shiny colored with distinct zone colony was picked and cultured in brain heart infusion (BHI) broth at 37 C and this culture was used for the extractions of cytoplasm and chromosomal DNA. The cytosolic fraction was used for the TK and TMPK enzyme assay while chromosomal DNA is used for amplification of TK and TMPK genes.16e18
2.2.
TK and TMPK enzyme assays
The enzyme activities were determined at 30 C using coupled spectrophotometric assay on a Cyber lab spectrophotometer USA. One unit of TK activity is defined as the amount of enzyme catalyzing the production of 1 mmol nucleoside monophosphate per minute whereas one unit of TMPK activity is defined as the amount of enzyme catalyzing the production of 1 mmol nucleoside diphosphate per minute. The kinetic parameters Km and Vmax were evaluated from HaneseWoolf plot ([S] vs [S/V]). Protein concentrations in all steps were determined by Bradford 1976 method.17e19
2.3. Cloning and expression of TMP kinase and TK genes from S. aureus ATCC12600 TMPK and TK genes were PCR amplified from the chromosomal DNA following the primers and conditions mentioned in Table 1.3 Thus, amplified products of TK and TMP kinase were purified with NP-PCR purification kit, Taurus Scientific, USA and were sequenced by dye terminating method at MWG Biotech India Ltd and the sequences were deposited at GenBank www.ncbi.nlm.nih.gov. The cloning of TK and TMPK genes were carried out as described earlier.19 The TK and TMPK genes were expressed using 1 mM IPTG from clones HTK and HTM respectively and pure rTK and rTMPK were obtained from respective clones were analyzed and characterized.17e19
Table 1 e PCR primers used in this study and the product size amplified from the chromosomal DNA isolated from S. aureus ATCC12600. Primers
Gene name
Forward HTKF 50 CATGTATGAAACTTAC30 Reverse HTKR 50 CGCGCGACAACGTG
TK
TMH1 forward 50 CATCTCAGCTTTTATAAC30 TMH2 reverse 50 GATGATAGTTTGATACGTG3’
TMPK
PCR conditions
Size of the PCR product (Kb)
Initial denaturation 94 C for 10 min, and for 40 cycles 94 C for 60 s, 42.1 C for 45 s, 72 C for 60 s and final extension at 72 C for 10 min Initial denaturation 94 C for 10 min, and for 40 cycles contain 94 C for 50 s of denaturation, 42.1 C for 45 s of annealing, 72 C for 60 s of extension and final elongation at 72 C for 10 min
0.55
0.58
91
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Table 2 e Kinetics of thymidine monophosphate kinase. S. No. 1. 2.
Source of TMP kinase
Enzyme activity (mMNADH/ml/min)
Vmax (mMNADH/mg/min)
Km (mM TMP)
Cytosolic fraction of S. aureus ATCC12600 Purified rTMP kinase from clone HTM clone.
0.43 0.02 0.52 0.02
32.3 1.5 31.33 1.5
0.85 0.03 1.33 0.04
Values are the mean SD from three determinations.
Table 3 e Kinetics of thymidine kinase. S. No. 1. 2.
Source of TK
Enzyme activity (mM NADH/ml/min)
Vmax (mM NADH/mg/min)
Km (mM thymidine)
Cytosolic fraction of S. aureus ATCC12600 Purified rTK kinase from clone HTK.
6.1 0.26 5.2 0.2
208 3 206.7 1.5
20.83 1.25 18.67 1.5
Values are the mean SD from three determinations.
Fig. 1 e Cloning of Staphylococcus aureus TMPK and TK genes A. PCR amplification of TMPK gene, lane M molecular size marker obtained from Bangalore Genei Pvt. Ltd, lane 1 and lane 2 PCR amplified products. B. PCR amplification of TK gene lane M molecular size marker obtained from Bangalore Genei Pvt. Ltd, lane 1 and 2 PCR amplified products. C. Schematic representation of pQE 30 plasmid vector. D. SDS-PAGE (10%) under reducing conditions of recombinant TMPK and TK from HTM (1) and HTK (2) clones respectively. (1) lane M molecular weight marker obtained from Bangalore Genei Pvt Ltd. Lane 1 uninduced HTM clone cytosolic fraction, lanes 2 and 3 induced cytosolic fraction of HTM clone with 1 mM IPTG. Lane 4 flow through collected after passing induced lysate through nickel metal chelate chromatographic column. Lane 5 recombinant TMPK eluted from nickel metal chelate column. (2) lane M molecular weight marker obtained from Bangalore Genei Pvt Ltd. Lanes 1 and 2 induced cytosolic fraction of HTK clone with 1 mM IPTG. Lane 3 uninduced HTK clone cytosolic fraction and lane 4 recombinant TK eluted from nickel metal chelate column.
92
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Fig. 2 e Multiple sequence alignment of TK of S. aureus with other bacteria and Homo sapiens. The highly conserved regions are indicated in mustard color, while partially in violet color. A. Multiple sequence alignment of S. aureus TK with bacteria and Homo sapiens TK sequences B. Sequence alignment of S. aureus and human TK sequences. Yellow and purple color sequences highlight the KEN and sequence in humans and absent in S. aureus.
2.4.
In silico analysis of TK and TMPK
TK and TMPK annotated protein sequences of S. aureus ATCC 12600 were analyzed by using the Internet available free softwares e NCBI BLAST, Bio-edit, Mega 4.1 and Clustal X.20e22 The translated TK and TMPK protein sequences were submitted to BLAST-P for similarity searching to find out its homologs.20 Pairwise and multiple sequence alignment were performed using Clustal X.21 The phylogenetic tree produced by the multiple sequence alignment was analyzed by using MEGA 4.1.22 The protein sequences were scanned against Pfam database to identify the conserved domains and family information of the proteins.23 The TK and TMPK structures of S. aureus were retrieved from (PDB IDs: 3E2I and 4DWJ) and were superimposed with human TK and TMPK (PDB IDs: 1XBT and 2XX3) using MATRAS program.24 The extent of homology between the structures was represented by respective RMSD values.
3.
Results and discussion
The TK and TMPK which are prominent enzymes involved in the formation of dTMP and dTDP respectively play critical role
Fig. 3 e Superimposed structures of S. aureus TK structure (PDB ID: 3E2I) (green) with human TK (PDB ID: 1XBT) (blue) were generated by using MATRAS program.
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Fig. 4 e A. Multiple sequence alignment of S. aureus TMPK with bacteria and Homo sapiens TMPK, highly conserved regions are indicated by yellow and violet. B. Sequence alignment of S. aureus and human TMPK sequences showing P-loop and LIDregions.
in the proliferation and pathogenesis of S. aureus in the human host especially in relapsed episodes and in SCV.4e6 TMPK is an enzyme which is in junction between de novo biosynthesis and salvage pathway and therefore, obtains substrates from both the pathways. The enzyme kinetics results of TK and TMPK (Tables 2 and 3) indicated that these enzymes are actively present in this pathogen (Supplementary Figs. 1 and 2). The TMPK and TK genes in the clones HTM and HTK respectively were confirmed by PCR using the primers mentioned in Table 1 and the insert in the clones were sequenced (GenBank accession numbers FJ415069 and FJ232923). The pure recombinant proteins eluted from nickel metal chelate agarose column (Bangalore Genei Pvt Ltd) exhibited single band in SDS-PAGE (10%) with a molecular weights of 21 kDa and 20 kDa respectively17 (Fig. 1). The structural superimposition results of S. aureus TK and TMPK and human TK and TMPK structures24 indicated RMSD
values of 0.913 A and 1.336 A respectively showing close homology between the structures (Figs. 3 and 5). However, TMPK structure of S. aureus exhibited typical characteristics of a class II enzyme, containing a G at position x1 of the P-loop whereas R is present in human TMPK and a series of basic residues (R 141, R 147, R 151 and K 144) in the LID region of S. aureus TMPK. Additionally, significant differences in the TMP site of S. aureus TMPK compared to other bacterial TMPKs and human TMPK have been identified.11,25,26 Also, human TMPK selectively phosphorylates the D enantiomer of dTMP and its analogs26 (Fig. 4A and B). This enantioselectivity of nucleosideactivating enzymes most likely have a strong impact on the efficacy and specificity of new antimicrobial agents. Human TK has very close homology with the TK of S. aureus ATCC12600 however, (Fig. 2A and B) humanTK1 has a unique KEN box in the C terminal region which is the binding site for ubiquitin ligase and thereby degrades HTK via an ubiquitin proteasome pathway,15 which is distinctly absent in the S. aureus TK.
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Fig. 5 e Superimposed structures of S. aureus TMPK structure (PDB ID: 4DWJ) (magenta) with human TMPK (PDB ID: 2XX3) (yellow) were generated by using MATRAS programme.
4.
Conclusion
In the present study TMPK and TK genes of S. aureus ATCC12600 have been cloned, expressed and characterized. The TMPK and TK kinetics clearly indicated that TMPK and TK are highly active enzymes in this pathogen and showed very close structural similarities with human TMPK and TK. However, absence of KEN sequence in S. aureus TK aids in the proliferation of this bacteria and the distinct differences observed in the substrate enantioselectivity of human TMPK conclude that dTMP analogs having L specificity could be strong antimicrobial agents. These unique differences correlated with variations in functions probably explains the rapid proliferation of S. aureus in its human host and which can be very serious and life threatening with the infections caused by multi drug resistant strains of S. aureus.
Conflicts of interest All authors have none to declare.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jopr.2013.01.021.
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