Comparison of the Arylamine N-Acetyltransferase from Mycobacterium marinum and Mycobacterium tuberculosis

Abstract

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr --> Phe and MMNAT209, Met --> Thr, in which residues within 6 A of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.

Probing the architecture of the Mycobacterium marinum arylamine N-acetyltransferase active site

“…If a similar reaction occurs within the MMNAT binding pocket, this could explain the inability to identify the previously proposed intermediate (Fig. 4B) and the higher efficiency with which HLZ is acetylated by NAT enzymes (Sikora et al, 2008;Fullam et al, 2009) compared with other aromatic hydrazines which have no comparable heterocyclic nitrogen e.g. isoniazid.…”

“…1) ) . Both enzymes show comparable substrate specificity profiles, especially in their selectivity to hydralazine (HLZ) as an acetyl group acceptor, with K m values of 60 μM in TBNAT (Sikora et al, 2008;Fullam et al, 2009) compared with 36 μM in MMNAT . Recently, the 3D structures of native MMNAT protein and its complex with CoA have been determined .…”

Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

“…The pure recombinant NAT enzyme from M. tuberculosis is relatively insoluble with maximum yields approaching approximately 2 mg per Liter of culture (Upton et al, 2001;Sikora et al, 2008;Lack et al, 2009;Fullam et al, 2009). In order to optimize the screening procedure, we have therefore used NAT proteins that have similar substrate specificities but are produced more abundantly as recombinant proteins at around 20 mg per liter of culture and that store well when pure (Westwood et al, 2005;Fullam et al, 2007Fullam et al, ,2009).…”

“…The pure recombinant NAT enzyme from M. tuberculosis is relatively insoluble with maximum yields approaching approximately 2 mg per Liter of culture (Upton et al, 2001;Sikora et al, 2008;Lack et al, 2009;Fullam et al, 2009). In order to optimize the screening procedure, we have therefore used NAT proteins that have similar substrate specificities but are produced more abundantly as recombinant proteins at around 20 mg per liter of culture and that store well when pure (Westwood et al, 2005;Fullam et al, 2007Fullam et al, ,2009). NAT isoenzymes have been characterized from a range of organisms and crystal structures have been obtained for the enzyme from Salmonella typhimurium (Sinclair et al, 2000), Pseudomonas aeruginosa (Westwood et al, 2005), the mycobacterial species Mycobacterium smegmatis (Sandy et al, 2002) and more recently from Mycobacterium marinum (Fullam et al, 2007;PDB code 2vfb), which is 74% identical to the enzyme from M. tuberculosis (Fullam et al, 2009).…”

“…In order to optimize the screening procedure, we have therefore used NAT proteins that have similar substrate specificities but are produced more abundantly as recombinant proteins at around 20 mg per liter of culture and that store well when pure (Westwood et al, 2005;Fullam et al, 2007Fullam et al, ,2009). NAT isoenzymes have been characterized from a range of organisms and crystal structures have been obtained for the enzyme from Salmonella typhimurium (Sinclair et al, 2000), Pseudomonas aeruginosa (Westwood et al, 2005), the mycobacterial species Mycobacterium smegmatis (Sandy et al, 2002) and more recently from Mycobacterium marinum (Fullam et al, 2007;PDB code 2vfb), which is 74% identical to the enzyme from M. tuberculosis (Fullam et al, 2009). A preliminary manual screen of a limited range of compounds using the NAT enzyme from M. smegmatis had already proved successful in identifying NAT inhibitors (Brooke et al, 2003b).…”