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Journal of Biochemistry Advance Access originally published online on September 30, 2008
Journal of Biochemistry 2008 144(6):709-715; doi:10.1093/jb/mvn124
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Establishment of Glutamine Synthetase of Mycobacterium smegmatis as a Protein Acetyltransferase utilizing Polyphenolic Acetates as the Acetyl Group Donors

Garima Gupta1, Anil Singh Baghel1, Seema Bansal1, Tapesh Kumar Tyagi1, Ranju Kumari1, Neeraj Kumar Saini1, Prija Ponnan1,2, Ajit Kumar1, Mridula Bose1, Daman Saluja3, Shamkant Anant Patkar4, Virinder Singh Parmar2 and Hanumantharao Guru Raj1,*

1Department of Biochemistry and Microbiology, V.P. Chest Institute; 2Department of Chemistry, University of Delhi, Delhi-1100 07; 3Dr B. R. Ambedkar Centre for Biomedical Research, India; and 4Department of Protein Biochemistry and Enzyme Design, Novozymes A/S, Smormosvej 25, DK 2880, Bagsvaerd, Denmark

*To whom correspondence should be addressed. Tel: +91-11-27667497, Fax: +91-11-27667420, E-mail: rajhg{at}yahoo.com

Received August 20, 2008; Accepted September 11, 2008


  Abstract

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have Mr 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.

Key Words: glutamine synthetase, Mycobacterium smegmatis, polyphenolic acetate, protein acetylation, transacetylase

Abbreviations: ABP, acetoxy benzopyran; 3-AC, 3-acetoxycoumarin; 4-AC, 4-acetoxycoumarin; 6-AMC, 6-acetoxy-4-methylcoumarin; 7-AMC, 7-acetoxy-4-methylcoumarin; CDNB, 1-chloro-2,4-dinitrobenzene; DAMC, 7,8-diacetoxy-4-methylcoumarin; GS, glutamine synthetase EC 6.3.1.2; GST, glutathione S-transferase; IPTG, iso thiopropyl galactoside; MSO, L-methionine-S-sulfoximine; MTAase, mycobacterial TAase; NOS, nitric oxide synthase; PA, polyphenolic acetates; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis; TAase, transacetylase


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