In Mycobacterium tuberculosis, the genes hsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) and nat (arylamine N-acetyltransferase) are essential for survival inside of host macrophages. These genes act as an operon and have been suggested to be involved in cholesterol metabolism. However, the role of NAT in this catabolic pathway has not been determined. In an effort to better understand the function of these proteins, we have expressed, purified and characterized TBNAT (NAT from M. tuberculosis) and HsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) from M. tuberculosis. Both proteins demonstrated remarkable heat stability with TBNAT and HsaD retaining >95% of their activity after incubation at 60 °C for 30 min. The first and second domains of TBNAT were demonstrated to be very important to the heat stability of the protein, as the transfer of these domains caused a dramatic reduction in the heat stability. The specific activity of TBNAT was tested against a broad range of acyl-CoA cofactors using hydralazine as a substrate. TBNAT was found to be able to utilize not just acetyl-CoA, but also n-propionyl-CoA and acetoacetyl-CoA, although at a lower rate. As propionyl-CoA is a product of cholesterol catabolism, we propose that NAT could have a role in the utilization of this important cofactor.
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March 2009
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Research Article|
February 11 2009
Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis
Nathan A. Lack;
Nathan A. Lack
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
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Akane Kawamura;
Akane Kawamura
†Summit PLC, 91 Milton Park, Abingdon OX14 4RY, U.K.
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Elizabeth Fullam;
Elizabeth Fullam
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
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Nicola Laurieri;
Nicola Laurieri
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
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Stacey Beard;
Stacey Beard
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
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Angela J. Russell;
Angela J. Russell
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
‡Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
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Dimitrios Evangelopoulos;
Dimitrios Evangelopoulos
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
§School of Biological and Chemical Sciences, Birkbeck College, University of London, Malet Street, London WC1E 7HX, U.K.
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Isaac Westwood;
Isaac Westwood
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
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Edith Sim
Edith Sim
1
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
October 07 2008
Revision Received:
November 13 2008
Accepted:
November 18 2008
Accepted Manuscript online:
November 18 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (2): 369–378.
Article history
Received:
October 07 2008
Revision Received:
November 13 2008
Accepted:
November 18 2008
Accepted Manuscript online:
November 18 2008
Citation
Nathan A. Lack, Akane Kawamura, Elizabeth Fullam, Nicola Laurieri, Stacey Beard, Angela J. Russell, Dimitrios Evangelopoulos, Isaac Westwood, Edith Sim; Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis. Biochem J 1 March 2009; 418 (2): 369–378. doi: https://doi.org/10.1042/BJ20082011
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