Rv0802c from Mycobacterium tuberculosis: the first structure of a succinyltransferase with the GNAT fold

Abstract

Gene rv0802c from Mycobacterium tuberculosis encodes a 218-amino-acid protein and is annotated as a hypothetical protein with homology to GCN5-related N-acetyltransferases. The structure of Rv0802c was determined in an unliganded form to 2.0 A resolution utilizing single-wavelength anomalous dispersion from a samarium soak that resulted in a single bound Sm(3+):citrate(2) complex. The structure confirms that Rv0802c exhibits the GCN5-related N-acetyltransferase fold and revealed a tetramer composed of a dimer of dimers with approximate 222 symmetry. In addition, a bound acetate ion indicated that Rv0802c may utilize a unique acyl donor for the family. The subsequent determination of the structure of Rv0802c in complex with succinyl-CoA to 2.3 A resolution suggests that Rv0802c is the first known GCN5-related N-acetyltransferase family member to utilize succinyl-CoA as a substrate.

Figure 1

Multiple sequence alignment of Rv0802c orthologs. Putative orthologs of Rv0802c (gi|15607942) from M. marinum (gi|183984857), Nocardioides sp. JS614 (gi|119717482), Arthrobacter sp. FB24 (gi|116669736), Kineococcus radiotolerans (gi|152968286) and Streptomyces sp. TP-A0584 (gi|78042205) were aligned using the program ClustalW (Thompson et al., 1994 ▶). Residues which are identical or similar in 75% of the sequences are shaded black and grey, respectively. Secondary-structural elements are indicated above the alignment and colored according to the ribbon diagrams in Fig. 3 ▶. Residues that are important in coordinating the succinyl moiety of SucCoA are indicated by red circles below the alignment. Residues which border a putative binding pocket for the second substrate are indicated by blue circles below the alignment. Values given in parentheses are the percentage identity to the M. tuberculosis Rv0802c sequence. The Streptomyces sp. TP-A0584 sequence is also termed GodH and is an acetyltransferase involved in goadsporin biosynthesis.

Figure 2

Derivatization of Rv0802c. (a) Final 2F _o − F _c electron density for the Sm³⁺:citrate² complex contoured at 2.5σ. (b) Stereoview of the coordination geometry of the Sm³⁺ ion.

Figure 3

Structure of Rv0802c. Ribbon diagrams for (a) an Rv0802c monomer, (b) a tetramer, (c) the A–B (C–D) dimer interface and (d) the A–D (B–C) dimer interface. The coordinates of the Rv0802c complex with SucCoA (sticks, colored by atom type) were used to make the diagram to illustrate the relative orientation of the four active sites and the flexible loop (α1–α2) which is not visible in the unliganded (acetate) structure. The presumed location of the active site is marked by an asterisk in (a).

Figure 4

The SucCoA-binding site. (a) F _o − F _c OMIT map contoured at 4σ for the acetate ion in the unliganded structure. Protein side chains which interact with the acetate are shown as sticks. (b) F _o − F _c OMIT map contoured at 2.5σ for SucCoA. (c) ChemDraw illustration of the interactions of Rv0802c with SucCoA. (d) Stereoview of the SucCoA-binding site of Rv0802c in the region of the succinyl moiety. OMIT maps were calculated after ligands had been removed, the coordinates had been randomized to produce an r.m.s.d. of 0.25 Å and the randomized ligand-omitted structure had undergone positional refinement.

Figure 5

Secondary binding groove. Residual electron density (final F _o − F _c contoured at 2.5σ) in a secondary binding groove adjacent to the SucCoA-binding site. Conserved binding residues are shown as sticks. SucCoA is shown as CPK spheres, with the succinyl moiety shown in brown C atoms and the acyl carbon marked with an asterisk.