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Specificity and mechanism of Acinetobacter baumanii nicotinamidase: implications for activation of the front-line tuberculosis drug pyrazinamide.

Nicotinamidase (EC 3.5.1.19) catalyzes hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD salvage pathway. This activity has a fortuitous medical benefit since the Mycobacterium tuberculosis enzyme converts the nicotinamide analogue prodrug pyrazinamide into the bacteriostatic pyrazinoic acid, hence the alternative name, pyrazinamidase (PncA). Pyrazinoic acid inhibits M. tuberculosis type I fatty acid synthase, represses mycolic acid biosynthesis, and appears to affect membrane energetics and acidification of the cytoplasm. It is active against semidormant tubercle bacilli and with rifampicin and isoniazid, forms the front-line tuberculosis treatment. 3] Though studies of PncA have revealed aspects of its structure and biochemical activity there are no structural data on how the enzyme binds and processes physiological ligands. Highresolution crystal structures of Acinetobacter baumanii PncA (AbPncA) complexed with nicotinic acid and pyrazinoic acid now provide direct evidence for the interactions that govern the specificity and mechanism, and of how a valued antibacterial agent is activated. Recombinant AbPncA was prepared, the dimeric, colorless enzyme was purified in high yield and its kinetic properties determined. With pyrazinamide as the substrate the following values were obtained; KM = 106.9 mm, Vmax = 62.8 nmol min , kcat. = 3.1 min , specific activity 132 mmmin 1 mg . These values are comparable to literature values, for example, the specific activity of M. tuberculosis PncA (MtPncA) with pyrazinamide is 82 mmmin 1 mg . Two crystal forms (I and II) were obtained with nicotinic and pyrazinoic acid, respectively, and the structures determined. PncA is a divalent cation-dependent enzyme and activity has been reported with Fe, Mn, and Zn ions. As expected, metal ions were observed in the structures. Inductively coupled plasma-atomic emission spectrometry (ICP-OES) identified that recombinant AbPncA contained Fe and Zn ions in an approximate 1:1 ratio with a trace of Mn present. However, anomalous dispersion measurements are consistent with a higher occupancy of Zn at the active site and the crystallographic models contain that cation. We refer to Zn ions in discussion but judge it likely that AbPncA functions in the presence of different divalent cations. (Experimental details, including enzyme activity and metal-ion identification, together with sequence alignments, and additional figures are given as Supporting Information Figure S1–S8). Crystals were obtained in the presence of cacodylate buffer and form II shows dimethylarsinoyl-modified Cys159 in the active site, an artifact of crystallization (Supporting Information, Figure S1, S2). The steric hindrance of this modification precludes full occupancy of pyrazinoic acid such that the final refinement was performed with occupancy 0.8 for pyrazinoic acid, 0.2 for the modified cysteine. Crystal form I has two molecules, form II a single molecule in the asymmetric unit, respectively, with a root-mean-square deviation (r.m.s.d.) derived from least-squares fit of Ca atoms of these three molecules of 0.2 . The structures and the interactions formed by ligands within the active sites are essentially identical and we concentrate on form I, a 1.65 resolution structure with full occupancy ligand (Supporting Information, Figure S3). About 60% of residues form elements of secondary structure, these are eight a-helices and nine b-strands (Figure 1, and Supporting Information, Figure S4). The core of the subunit is a parallel b-sheet of strands 1, 2, 5–9. Three helices (a5, a6, a7) lie on one side of the sheet, with a2 placed against the other. A subdomain is placed at