Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600
Background: Staphylococcus aureus possesses complete tricarboxylic acid (TCA) cycle. Succinate dehydrogenase (SDH) links TCA cycle with electron transport chain and could therefore be an ideal target in the development of new antimicrobials. Hence, present study is aimed at characterization of SDH...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2013-04-01
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| Series: | Journal of Clinical and Scientific Research |
| Subjects: | |
| Online Access: | http://svimstpt.ap.nic.in/jcsr/apr-june%202013_files/apr-jun13/oa%203.pdf |
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| author | V. Swarupa S. Yeswanth U.V. Prasad D. Vasu K. Venkatesh L. Srikanth P. Anitha C. Manasa P.Santhosh Kumar G. Sowjenya Y. Nandakumar Abhijit Chaudhary P.V.G.K. Sarma |
| author_facet | V. Swarupa S. Yeswanth U.V. Prasad D. Vasu K. Venkatesh L. Srikanth P. Anitha C. Manasa P.Santhosh Kumar G. Sowjenya Y. Nandakumar Abhijit Chaudhary P.V.G.K. Sarma |
| author_sort | V. Swarupa |
| collection | DOAJ |
| description | Background: Staphylococcus aureus possesses complete tricarboxylic acid (TCA) cycle. Succinate dehydrogenase
(SDH) links TCA cycle with electron transport chain and could therefore be an ideal target in the development of new
antimicrobials. Hence, present study is aimed at characterization of SDH flavoprotein from Staphylococcus aureus ATCC 12600.
Methods: Staphylococcus aureus ATCC 12600 was grown in brain heart infusion (BHI) broth and from the membrane
fraction SDH was isolated and purified using diethyl aminoethyl (DEAE) cellulose. The kinetic parameters Michaelis
constant (KM), maximal velocity (Vmax) and rate constant (kcat) for both native (SDH) and recombinant succinate dehydrogenase (rSDH) enzyme flavoproteins were determined through Hanes-Woolf plot. The SDH flavoprotein gene was
amplified using polymerase chain reaction (PCR) and was cloned in pQE30 vector and expressed in DH5 strain of
Escherichia coli. The molecular weights of native and rSDH flavoproteins were determined by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE).
Results: SDH flavoprotein was purified from the membrane fraction of S.aureus and the present methodology gave 40
folds purification with molecular weight of 66kDa. The Vmax, KM and kcat of SDH flavoprotein are 199±1 μM/mg/min,
143.5±0.1 μM and 995±4/min. The pure recombinant SDH flavoprotein exhibited similar properties with that of native
SDH flavoprotein.
Conclusions: In multidrug-resistant strains of Staphylococcus aureus up regulation of SDH was observed favouring increased biofilm formation which is one of the key pathogenic factor and in view of the differences observed in the kinetics of Staphylococcus aureus SDH flavoprotein with other bacteria this enzyme is probably regarded as an ideal drug target in the development of new antimicrobials. |
| first_indexed | 2024-04-11T16:11:24Z |
| format | Article |
| id | doaj.art-fee668edc84440d6bcd668120bfd02b1 |
| institution | Directory Open Access Journal |
| issn | 2277-5706 2277-8357 |
| language | English |
| last_indexed | 2024-04-11T16:11:24Z |
| publishDate | 2013-04-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Journal of Clinical and Scientific Research |
| spelling | doaj.art-fee668edc84440d6bcd668120bfd02b12022-12-22T04:14:41ZengWolters Kluwer Medknow PublicationsJournal of Clinical and Scientific Research2277-57062277-83572013-04-01228187Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600V. Swarupa0S. Yeswanth1 U.V. Prasad2 D. Vasu3 K. Venkatesh4 L. Srikanth5 P. Anitha6 C. Manasa7 P.Santhosh Kumar8 G. Sowjenya9Y. Nandakumar10Abhijit Chaudhary11 P.V.G.K. Sarma12Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati Department of Zoology, Sri Venkateswara University, Tirupati Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, TirupatiDepartment of Biotechnology, Sri Venkateswara Institute of Medical Sciences, TirupatiBackground: Staphylococcus aureus possesses complete tricarboxylic acid (TCA) cycle. Succinate dehydrogenase (SDH) links TCA cycle with electron transport chain and could therefore be an ideal target in the development of new antimicrobials. Hence, present study is aimed at characterization of SDH flavoprotein from Staphylococcus aureus ATCC 12600. Methods: Staphylococcus aureus ATCC 12600 was grown in brain heart infusion (BHI) broth and from the membrane fraction SDH was isolated and purified using diethyl aminoethyl (DEAE) cellulose. The kinetic parameters Michaelis constant (KM), maximal velocity (Vmax) and rate constant (kcat) for both native (SDH) and recombinant succinate dehydrogenase (rSDH) enzyme flavoproteins were determined through Hanes-Woolf plot. The SDH flavoprotein gene was amplified using polymerase chain reaction (PCR) and was cloned in pQE30 vector and expressed in DH5 strain of Escherichia coli. The molecular weights of native and rSDH flavoproteins were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Results: SDH flavoprotein was purified from the membrane fraction of S.aureus and the present methodology gave 40 folds purification with molecular weight of 66kDa. The Vmax, KM and kcat of SDH flavoprotein are 199±1 μM/mg/min, 143.5±0.1 μM and 995±4/min. The pure recombinant SDH flavoprotein exhibited similar properties with that of native SDH flavoprotein. Conclusions: In multidrug-resistant strains of Staphylococcus aureus up regulation of SDH was observed favouring increased biofilm formation which is one of the key pathogenic factor and in view of the differences observed in the kinetics of Staphylococcus aureus SDH flavoprotein with other bacteria this enzyme is probably regarded as an ideal drug target in the development of new antimicrobials.http://svimstpt.ap.nic.in/jcsr/apr-june%202013_files/apr-jun13/oa%203.pdfSuccinate dehydrogenase flavoproteinKMVmaxStaphylococcus aureus |
| spellingShingle | V. Swarupa S. Yeswanth U.V. Prasad D. Vasu K. Venkatesh L. Srikanth P. Anitha C. Manasa P.Santhosh Kumar G. Sowjenya Y. Nandakumar Abhijit Chaudhary P.V.G.K. Sarma Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 Journal of Clinical and Scientific Research Succinate dehydrogenase flavoprotein KM Vmax Staphylococcus aureus |
| title | Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 |
| title_full | Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 |
| title_fullStr | Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 |
| title_full_unstemmed | Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 |
| title_short | Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600 |
| title_sort | characterization of succinate dehydrogenase flavoprotein from staphylococcus aureus atcc 12600 |
| topic | Succinate dehydrogenase flavoprotein KM Vmax Staphylococcus aureus |
| url | http://svimstpt.ap.nic.in/jcsr/apr-june%202013_files/apr-jun13/oa%203.pdf |
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