title: Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis.
abstract: 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 degrees 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.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX13QT, UK.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Lack
authors.forename: Akane
authors.initials: A
authors.lastname: Kawamura
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fullam
authors.forename: Nicola
authors.initials: N
authors.lastname: Laurieri
authors.forename: Stacey
authors.initials: S
authors.lastname: Beard
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Russell
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Evangelopoulos
authors.forename: Isaac
authors.initials: I
authors.lastname: Westwood
authors.forename: Edith
authors.initials: E
authors.lastname: Sim
mesh_terms: D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics; D050296:Microbial Viability / Q000235:genetics; D008954:Models, Biological; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002784:Cholesterol; D001191:Arylamine N-Acetyltransferase; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis; D003065:Coenzyme A