Recombinant Expression of a Functional Myo-Inositol-1-Phosphate Synthase (MIPS) in Mycobacterium smegmatis

详细信息    查看全文

• 作者：Xinyi Huang ; Marcy Hernick
• 关键词：Myo ; inositol ; 1 ; phosphate synthase (MIPS) ; Mycobacteria ; Recombinant expression ; Mycobacterium smegmatis ; Mycobacterium tuberculosis
• 刊名：The Protein Journal
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：34
• 期：5
• 页码：380-390
• 全文大小：1,883 KB
• 参考文献：1.Michell RH (2008) Inositol derivatives: evolution and functions. Nat Rev Mol Cell Biol 9:151-61CrossRef
  2.Huang X, Hernick M (2013) In: Rocha H, Cardoso M (eds) Inositol derivatives in Mycobacterium tuberculosis: function, biosynthesis and therapeutic implications. Nova Science Publishers, Hauppauge
  3.Morita YS, Fukuda T, Sena CB, Yamaryo-Botte Y, McConville MJ, Kinoshita T (2011) Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms. Biochim Biophys Acta 1810:630-41CrossRef
  4.Mishra AK, Driessen NN, Appelmelk BJ, Besra GS (2011) Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host-pathogen interaction. FEMS Microbiol Rev 35:1126-157CrossRef
  5.Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev 72:471-94CrossRef
  6.Hernick M (2013) Mycothiol, a target for potentiation of rifampin and other antibiotics against M. tuberculosis. Expert Rev Anti-Infect Ther 11:49-7CrossRef
  7.Bettaney KE, Sukumar P, Hussain R, Siligardi G, Henderson PJ, Patching SG (2012) A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis. Mol Membr Biol 30:3-4CrossRef
  8.Movahedzadeh F, Smith DA, Norman RA, Dinadayala P, Murray-Rust J, Russell DG, Kendall SL, Rison SC, McAlister MS, Bancroft GJ, McDonald NQ, Daffe M, Av-Gay Y, Stoker NG (2004) The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence. Mol Microbiol 51:1003-014CrossRef
  9.Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-44CrossRef
  10.Bachhawat N, Mande SC (1999) Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme. J Mol Biol 291:531-36CrossRef
  11.Bachhawat N, Mande SC (2000) Complex evolution of the inositol-1-phosphate synthase gene among archaea and eubacteria. Trends Genet 16:111-13CrossRef
  12.Majumder AL, Chatterjee A, Ghosh Dastidar K, Majee M (2003) Diversification and evolution of l -myo-inositol 1-phosphate synthase. FEBS Lett 553:3-0CrossRef
  13.Li Y, Chen Z, Li X, Zhang H, Huang Q, Zhang Y, Xu S (2007) Inositol-1-phosphate synthetase mRNA as a new target for antisense inhibition of Mycobacterium tuberculosis. J Biotechnol 128:726-34CrossRef
  14.Smith D, Hansch H, Bancroft G, Ehlers S (1997) T-cell-independent granuloma formation in response to Mycobacterium avium: role of tumour necrosis factor-alpha and interferon-gamma. Immunology 92:413-21CrossRef
  15.Hansch HC, Smith DA, Mielke ME, Hahn H, Bancroft GJ, Ehlers S (1996) Mechanisms of granuloma formation in murine Mycobacterium avium infection: the contribution of CD4+ T cells. Int Immunol 8:1299-310CrossRef
  16.Norman RA, McAlister MS, Murray-Rust J, Movahedzadeh F, Stoker NG, McDonald NQ (2002) Crystal structure of inositol 1-phosphate synthase from Mycobacterium tuberculosis, a key enzyme in phosphatidylinositol synthesis. Structure?10:393-02CrossRef
  17.Chen L, Zhou C, Yang H, Roberts MF (2000) Inositol-1-phosphate synthase from Archaeoglobus fulgidus Is a class II aldolase. Biochemistry 39:12415-2423CrossRef
  18.Escamilla JE, Contreras M, Martinez A, Zentella-Pina M (1982) l -myoinositol-1-phosphate synthase from Neurospora crassa: purification to homogeneity and partial characterization. Arch Biochem Biophys 218:275-85CrossRef
  19.Neelon K, Wang Y, Stec B, Roberts MF (2005) Probing the mechanism of the Archaeoglobus fulgidus inositol-1-phosphate synthase. J Biol Chem 280:11475-1482CrossRef
  20.Stieglitz KA, Yang H, Roberts MF, Stec B (2005) Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-Inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus. Biochemistry 44:213-24CrossRef
  21.Geiger JH, Jin X (2006) In: Majumder AL, Biswas BB (eds) The structure and mechanism of myo-inositol-1-phosphate synthase. Springer, USCrossRef
  22.Stieglitz KA, Yang H, Roberts MF, Stec B (2005) Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus. Biochemistry 44:213-24CrossRef
  23.Jin X, Geiger JH (2003) Structures of NAD(+)-and NADH-bound 1-l-myo-inositol 1-phosphate
• 作者单位：Xinyi Huang (1)
  Marcy Hernick (1) (2)
  
  1. Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, USA
  2. Department of Pharmaceutical Sciences, Appalachian College of Pharmacy, Oakwood, VA, 24631, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Bioorganic Chemistry
  Biochemistry
  Organic Chemistry
  Animal Anatomy, Morphology and Histology
  
• 出版者：Springer Netherlands
• ISSN：1573-4943

文摘

Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production—the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD+ cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc24517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis. Keywords Myo-inositol-1-phosphate synthase (MIPS) Mycobacteria Recombinant expression Mycobacterium smegmatis Mycobacterium tuberculosis