protein structure communications
Structure of Mesorhizobium loti arylamine N-acetyltransferase 1
aLaboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, England, bCNRS-UMR 7000, Faculté de Médecine Pitié-Salpêtrière, 105 Boulevard de l'Hôpital, 75013 Paris, France, cDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, England, and dUFR de Biochimie, Université Denis Diderot-Paris 7, 75005 Paris, France
*Correspondence e-mail: edith.sim@pharm.ox.ac.uk
The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site.
Keywords: arylamine N-acetyltransferase 1.
3D view: 1v09
PDB reference: arylamine N-acetyltransferase 1, 1v09, r1v09sf
1. Introduction
The arylamine N-acetyltransferase (NAT) family of phase II enzymes catalyse the acetylation of primary arylamines, arylhydrazines and N-hydroxylated metabolites (Riddle & Jencks, 1971; Weber & Hein, 1985; Evans, 1989; Delomenie et al., 2001). Initially identified in humans through their ability to inactivate the anti-tuberculosis therapeutic isoniazid, NAT-encoding sequences have now been identified in a broad range of eukaryotic and prokaryotic species (Pompeo et al., 2002). To date, X-ray crystallographic structures of three NAT paralogues have been reported, all of which possess a highly conserved three-domain fold whose active sites contain a triad of residues (Cys-His-Asp) proposed to catalyse substrate acetylation (Sinclair et al., 2000).
Mesorhizobium loti is a nitrogen-fixing rhizobacterium that lives in symbiosis with leguminous plants (Kaneko et al., 2000). Recent bioinformatics analysis of the M. loti genome revealed two NAT isoforms, NAT1 and NAT2, and was the first example of a eubacterial species with multiple NAT isoforms (Rodrigues-Lima & Dupret, 2002). In this paper, we report the 2 Å X-ray crystallographic structure of M. loti NAT1, a comparison with other NAT structures and a discussion of the implications for M. loti NAT2.
2. Experimental
The M. loti NAT1 open-reading frame was cloned, expressed and purified from M. loti MAFF303099 strain. Details of cloning, expression and purification will be published elsewhere.
Pure recombinant protein (in 20 mM Tris–HCl pH 7.5, 1 mM EDTA, 1 mM DTT) was concentrated to 10 mg ml−1 using Amicon ultracentrifugation concentrators (Millipore, Watford, Hertfordshire). The crystals described in this paper were grown at 292 K using the sitting-drop vapour-diffusion technique, with initial conditions formed by mixing equal volumes (1 µl) of the concentrated M. loti NAT1 solution with mother liquor [0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5]. Typically, crystals appeared after 2–5 d.
Crystals cryoprotected in paraffin oil were frozen at 100 K and diffraction data were collected at beamline ID14-2 at the European Synchrotron Radiation Facility, Grenoble, France. Data were indexed, integrated and scaled using the programs MOSFLM and SCALA (Collaborative Computational Project, Number 4, 1994). Initial phases were determined by with the program AMoRe using NAT from Mycobacterium smegmatis (PDB code 1gx3 ) as a search model. Initial model building was carried out using a combination of automatic rebuilding programs (ARP/wARP and FFFEAR; Collaborative Computational Project, Number 4, 1994) and manual rebuilding using the program O (Jones et al., 1991). Subsequent involved iterative manual rebuilding and using the program REFMAC5 (Collaborative Computational Project, Number 4, 1994). A final step was to solvate the protein using the program ARP (Perrakis et al., 1999). The stereochemical quality of the final model was verified using the program PROCHECK (Laskowski et al., 1993). are presented in Table 1.
‡Rconv = , where Foh and Fch are the observed and calculated structure-factor amplitudes for reflection h. §Rfree is equivalent to Rconv, but is calculated using a 5% disjoint set of reflections excluded from the stages. |
3. Results and discussion
The M. loti NAT1 structure adopts the characteristic three-domain NAT fold in which the first two domains, a helical bundle and a β-barrel, are aligned to allow three residues (Cys73, His112 and Asp127, M. loti NAT1 numbering) to form a catalytic triad (Fig. 1). This three-residue structural motif is well documented in NATs and is similar to that found in the structurally related cysteine protease superfamily (Sinclair et al., 2000). The relative geometry of these catalytic residues is absolutely conserved between M. loti NAT1 and other NAT structures. The sequence and structural homology within the active site of M. loti NAT1 and other NATs is significantly higher than the global sequence identity, which ranges between 34 and 41%, and suggests that the different NAT isoforms share a common enzymatic mechanism. The third domain is linked to the second via an interdomain helix (residues 202–207), whilst the interface formed between domains II and III and the lid region together form a substantial active-site cleft. This structure extends the phylogenetic range of species over which NAT folds have been observed and supports the prediction of a conserved fold for bacterial NAT orthologues (Payton et al., 2001; Westwood et al., 2005).
Comparisons between NAT structures have previously revealed minor conformational differences resulting from both single-residue insertions/deletions that are accommodated within loop regions of the structure and from mobile loop regions linking more stable secondary-structural elements (Sinclair et al., 2000; Sandy et al., 2002; Westwood et al., 2005). These result in little perturbation to the protein fold and as such are not considered significant. The superposition of M. loti NAT1 with other NAT structures reveals a similar pattern of structural conservation (Fig. 1). The largest and potentially most significant deviation centres on the loop between β3 and β4 (approximately residue 103). Although the conformation of this loop differs between all published NAT structures, this is the only example where accessibility of the active site is potentially reduced owing to the conformation of the loop (Fig. 1). Little is known about the interactions between NATs and their substrates/cofactors and so the effect, if any, of such a conformation on the enzymatic character of M. loti NAT1 remains to be seen.
Despite sharing moderate to low levels of sequence identity, the high level of structural homology between NAT structures suggests that M. loti NAT2 will adopt a similar fold. Mapping sequence conservation between the two M. loti NAT paralogues onto the NAT1 structure reveals that with the exception of the active-site core, there are no extensive regions of residue conservation (data not shown). This may reflect a differentiation of cellular role and/or location from a common ancestral protein or simply a lack of evolutionary pressure on conservation away from the active site. In human cells, NAT isoforms exhibit overlapping but distinct substrate selectivity and cellular location profiles (Sim et al., 2000). If the M. loti paralogues represent a similar scenario, then the unusually low levels of residue conservation may reflect more divergent enzymatic roles.
In this communication, we have presented the X-ray crystallographic structure of M. loti NAT1. Despite low sequence similarity, M. loti NAT1 shares the well conserved NAT fold and highlights the sensitive nature of the profile-based search methods which first identified the sequence as encoding a NAT (Rodrigues-Lima & Dupret, 2002). The M. loti NAT1 structure adds to the growing number of published NAT structures and will complement current work aimed at identifying bacterial NAT antagonists.
Acknowledgements
We are grateful to the Wellcome Trust for continued financial support. We also thank Association pour la Recherche sur le Cancer (ARC), Association Française contre les Myopathies and Rétina France for financial support. We gratefully acknowledge the help of the beamline scientists at beamline ID14-2 at the ESRF, Grenoble and also thank Isaac Westwood for helpful discussions. This work would not have been possible without the support of the Franco–British Alliance Partnership Programme (No. PN 03.066).
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