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Review
. 2008 Jul;9(6):510-9.
doi: 10.2174/138920008784892100.

Arylamine N-acetyltransferases in mycobacteria

Edith Sim  1 , James Sandy, Dimitrios Evangelopoulos, Elizabeth Fullam, Sanjib Bhakta, Isaac Westwood, Anna Krylova, Nathan Lack, Martin Noble
Affiliations
Free PMC article
Review

Arylamine N-acetyltransferases in mycobacteria

Edith Sim et al. Curr Drug Metab. 2008 Jul.
Free PMC article

Abstract

Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure.

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Figures

Fig. (1). Comparison of the amino acid sequences of NATs from mycobacteria.
Fig. (1). Comparison of the amino acid sequences of NATs from mycobacteria.
A schematic diagram to show the amino acids which differ between MMNAT and TBNAT based on a ClustalW alignment (http://www.ebi.ac.uk/clustalw) and Espript [63]. The amino acids indicated above the bar are TBNAT residues and the amino acids indicated below the bar are MMNAT residues. White lines indicate residues which are a conserved substitution, grey lines indicate residues which are semi-conserved, black lines indicate residues which are nonconservative and the dashes indicate deletions since MMNAT is shorter than TBNAT. The numbering is based on the TBNAT sequence. Amino acids are indicated by single letters.
Fig. (2). Comparison of the operon organization of mycobacterial species.
Fig. (2). Comparison of the operon organization of mycobacterial species.
Operon analysis was carried out using the method described in [21] and also [31]. Genes have been named as they are annotated in the relevant genomes: nat = arylamine N-acetyltransferase; pseudo = pseudogene; hsa gene cluster is named as described in [21] and [26]; aspB is an aspartate aminotransferase; araC is homologous to the Escherichia coli transcription regulator [64]; deam/reduct refers to deaminase/reductase gene product; dh, dh1 and dh2 correspond to homologues of dehydrogenase gene products; dhfr = dihydrofolate reductase. The nat genes in M. tuberculosis, M. bovis and M. smegmatis, and also hsaD and hsaC from M. tuberculosis and M. bovis encode proteins where enzyme activity has been confirmed. All of the open reading frames encode for putative proteins. See http://genolist.pasteur.fr/TubercuList/, http://www.tigr.org, http://www.sanger.ac.uk/Projects/Microbes/
Fig. (3). Overlay of the crystal structures of NATs from bacteria.
Fig. (3). Overlay of the crystal structures of NATs from bacteria.
NATs from M. smegmatis (pdb code 1gx3), S. typhimurium (pdb code 1e2t), P. aeruginosa (pdb code 1w4f) and M. loti NAT1 (pdb code 2bsz) are shown superimposed, each in a different grey tone. The active site triad residues are shown in ball and stick representation and are indicated by arrows. After [13].
Fig. (4). Interactions of isoniazid with the active site of NAT from <i>M. smegmatis.</i>
Fig. (4). Interactions of isoniazid with the active site of NAT from M. smegmatis.
The interactions are shown using Ligplot analysis [65]. Inh1 (E) indicates isoniazid. The residues are indicated by their numbers in M. smegmatis NAT. After [7]. An identical Ligplot for M. marinum NAT is found in [43].
Fig. (5). Comparison of the interaction of CoA with NAT from <i>M. marinum</i> and human NAT2.
Fig. (5). Comparison of the interaction of CoA with NAT from M. marinum and human NAT2.
Molecular surface representations of M. marinum NAT with CoA bound (pdb 2vfc) (A) with human NAT2 with CoA bound (pdb 2pfr) (B). The CoA is shown in ball and stick representation. Ribbon representation of the binding of M marinum NAT (MMNAT) with CoA bound (dark ribbon) compared with CoA bound to human NAT2(hNAT2) (light ribbon). The structures have been overlaid and the CoA molecules are shown in ball and stick representation, as are the residues of the active site triad (Cys indicated by an arrow) (C). Frame (D) shows the location of isoniazid in the active site, in relation to the position of CoA in the structure of M. marinum NAT. Isoniazid and CoA are shown in ball and stick representation and the active site resides are just visible, with Cys being indicated by an arrow. After [43].
Fig. (6). The reactions leading to the synthesis of Geldanamycin by amide ring closure.
Fig. (6). The reactions leading to the synthesis of Geldanamycin by amide ring closure.
A. The series of biosynthetic reactions in Streptomyces leading to biosynthesis of the benzoquinone ansamycin geldanamycin. B. the ring closure reaction catalysed by an amide synthetase is highlighted. (After [58,59]). AHBA is amino hydroxybenzoic acid. The amide synthetase is encoded by the gene gdmf. The earlier genes in the cluster are numbered alphabetically. PKS is polyketide synthetase.
Fig. (7). Alignment of putative Amide Synthetase protein sequences.
Fig. (7). Alignment of putative Amide Synthetase protein sequences.
The sequences of the putative amide synthetases are shown in comparison with human NAT1 (P50295) and NAT from Mycobacterium smegmatis (086309). The essential residues as indicated are after [61]. The amide synthetases listed are as follows: O52547|RIFF_AMYMD 3-amino-5-hydroxybenzoic acid synthase - Amycolatopsis mediterranei (Nocardia mediterranei)., A8M6D1|A8M6D1_9ACTO N-acetyltransferase - Salinispora arenicola , Q2PC63|Q2PC63_STRAH Putative amide synthase - Streptomyces achromogenes subsp. Rubradiris, Q84G21|Q84G21_STRHY GdmF - Streptomyces hygroscopicus, Q1L0S9|Q1L0S9_STRHY GelD - Streptomyces hygroscopicus subsp. Duamyceticus, A8LFZ8|A8LFZ8_9ACTO N-acetyltransferase - Frankia sp. EAN1pec., A4FDM9|A4FDM9_SACEN 3-amino-5-hydroxybenzoic acid synthase - Saccharopolyspora erythraea (strain NRRL 23338), Q8KUF7|Q8KUF7_ACTPA Amide synthase - Actinosynnema pretiosum subsp. auranticum., Q848C2|Q848C2_STRHY Amide synthase - Streptomyces hygroscopicus (sequence cropped @ N-term to remove cloning artifacts)., Q0PLZ9|Q0PLZ9_9ACTO Putative N-acetyltransferase - Kitasatospora putterlickiae., Q8GNN3|Q8GNN3_STRHY ShnN - Streptomyces hygroscopicus., Q1L0S3|Q1L0S3_STRHY NapF - Streptomyces hygroscopicus subsp. duamyceticus.
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