全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
PLOS ONE  2012 

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

DOI: 10.1371/journal.pone.0035998

Full-Text   Cite this paper   Add to My Lib

Abstract:

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.

References

[1]  Meister GE, Kanwar M, Ostermeier M (2009) Circular permutation of proteins. In: Lutz S, Bornscheuer U, editors. Protein Engineering Handbook: Wiley.
[2]  Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96: 11241–11246.
[3]  Graf R, Schachman HK (1996) Random circular permutation of genes and expressed polypeptide chains: Application of the method to the catalytic chains of aspartate transcarbamoylase. Proc Natl Acad Sci USA 93: 11591–11596.
[4]  Topell S, Hennecke J, Glockshuber R (1999) Circularly permuted variants of the green fluorescent protein. FEBS Lett 457: 283–289.
[5]  Iwakura M, Nakamura T, Yamane C, Maki K (2000) Systematic circular permutation of an entire protein reveals essential folding elements. Nat Struct Biol 7: 580–585.
[6]  Cheltsov AV, Barber MJ, Ferreira GC (2001) Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function. J Biol Chem 276: 19141–19149.
[7]  Qian Z, Lutz S (2005) Improving the catalytic activity of Candida antarctica lipase B by circular permutation. J Am Chem Soc 127: 13466–13467.
[8]  Qian Z, Fields CJ, Lutz S (2007) Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica. Chembiochem 8: 1989–1996.
[9]  Reitinger S, Yu Y, Wicki J, Ludwiczek M, D'Angelo I, et al. (2010) Circular permutation of Bacillus circulans xylanase: a kinetic and structural study. Biochemistry 49: 2464–2474.
[10]  Yu Y, Lutz S (2009) Improved triglyceride transesterification by circular permuted Candida antarctica lipase B. Biotechnol Bioeng 105: 44–50.
[11]  Qian Z, Horton JR, Cheng X, Lutz S (2009) Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation. J Mol Biol 393: 191–201.
[12]  Guntas G, Mitchell SF, Ostermeier M (2004) A molecular switch created by in vitro recombination of nonhomologous genes. Chem Biol 11: 1483–1487.
[13]  Ostermeier M (2005) Engineering allosteric protein switches by domain insertion. Protein Engineering Design and Selection 18: 359–364.
[14]  Fisher JF, Mobashery S (2009) Three decades of the class A beta-lactamase acyl-enzyme. Curr Protein Pept Sci 10: 401–407.
[15]  Hayes F, Hallet B, Cao Y (1997) Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase. J Biol Chem 272: 28833–28836.
[16]  Petrosino JF, Palzkill T (1996) Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase. J Bacteriol 178: 1821–1828.
[17]  Salverda ML, De Visser JA, Barlow M (2010) Natural evolution of TEM-1 beta-lactamase: experimental reconstruction and clinical relevance. FEMS Microbiol Rev 34: 1015–1036.
[18]  Osuna J, Pérez-Blancas A, Soberón X (2002) Improving a circularly permuted TEM-1 β-lactmase by directed evolution. Protein Eng 15: 463–470.
[19]  Guntas G, Mansell TJ, Kim JR, Ostermeier M (2005) Directed evolution of protein switches and their application to the creation of ligand-binding proteins. Proc Natl Acad Sci USA 102: 11224–11229.
[20]  Bosley AD, Ostermeier M (2005) Mathematical expressions useful in the construction, description and evaluation of protein libraries. Biomolecular Engineering 22: 57–61.
[21]  Minasov G, Wang X, Shoichet BK (2002) An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation. J Am Chem Soc 124: 5333–5340.
[22]  Ostermeier M, Nixon AE, Shim JH, Benkovic SJ (1999) Combinatorial protein engineering by incremental truncation. Proc Natl Acad Sci USA 96: 3562–3567.
[23]  Ostermeier M, Shim JH, Benkovic SJ (1999) A combinatorial approach to hybrid enzymes independent of DNA homology. Nat Biotechnol 17: 1205–1209.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133