Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis

Abstract

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R(free) of 0.228 and 0.263, respectively, at 2.3 A resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.

Figure 1

Dimer structure of BA NaMNAT (chain A in green and chain B in cyan). Secondary structures are labelled for chain A.

Figure 2

(a) Overlay of apo BA NaMNAT (PDB code 3dv2, green; 2qtm, blue), apo BS NaMNAT (1kam, yellow) and BS NaMNAT–NaAD complex (1kaq, red; NaAD is shown in stick representation and colored by element). Secondary structures, N- and C-termini are labelled. (b–d) The three regions with the greatest conformational flexibility. Each region is enlarged and rotated to show most clearly the differences in conformation between the proteins. All structures shown in (a) plus BA NaMNAT–NaAD (PDB code 2qtr; magenta) are included. The first and last residues of each region are labelled for BA.

Figure 3

Sequence alignment of NaNMATs from B. anthracis (BA), B. subtilis (BS), S. aureus (SA), E. coli (EC) and P. aeruginosa (PA). The structure of BA NaMNAT was used to generate the structural annotations. The alignment was performed with ClustalW (Chenna et al., 2003 ▶) and the figure was prepared with ESPript (Gouet et al., 1999 ▶)

Figure 4

Residues 43–51 in apo BA NaMNAT are shown in green with a 2F _o − F _c electron-density map at 1σ in blue. Part of a symmetry-related molecule is shown in yellow. Crystal packing between the two chains is obvious and could account for the stability of the loop 43–51. These residues are not visible in the BS apo structure, which does not have the same packing.