Biochemical and Biophysical Research Communications
Crystal structure of the nicotinamidase/pyrazinamidase PncA from Bacillus subtilis
Introduction
PncA is a nicotinamidase encoded by the pncA gene that functions in the NAD salvage pathway by converting nicotinamide (NAM) to nicotinic acid [[1], [2], [3]]. PncA acts as a key effector of processes related to mild stress-induced lifespan extension, vitamin B3/niacin metabolism, and calorie restriction [[4], [5], [6]]. In addition to these known biological functions, PncA is also a pyrazinamidase that is requred for the activation of the front-line antituberculosis drug pyrazinamide (PZA). Nicotinamidases/pyrazinamidases in Mycobacterium tuberculosis convert pyrazinamide into the active form of the drug, pyrazinoic acid [7]. PncA has also been found in many pathogenic microorganisms, such as Flavobacterium peregrinum, Staphylococcus aureus, and Saccharomyces cerevisiae [[8], [9], [10]]. PZA resistance is another important factor in the control of these bacteria [11]. PZA resistance is associated with mutations in the pncA gene [12,13]. Therefore, an understanding of the chemical mechanisms underlying the transition state of the reaction catalyzed by this enzyme is crucial for the development of new and effective agents to combat anti-PZA resistance in pathogenic bacteria.
PncA is a metalloenzyme, and the metal coordination site (MCS) is known to play a pivotal role in its enzymatic activity [14]. As shown in the study by Sheen et al., metal-depleted M. tuberculosis PncA exhibits different reactivation patterns that depend on the substituted metal ion [15]. These researchers also found that, among various metal ions, Co2+, Mn2+, Fe2+, and Zn2+ reactivated metal-depleted PncA [15,16]. Mutations in the active site or the metal-binding site affect PncA activity and even mutations located far from these sites decrease enzyme activity to some degree [13,[16], [17], [18], [19]]. The structures of PncA homologues in various organisms, such as M. tuberculosis [Protein Data Bank (PDB) entry 3PL1] [20], S. cerevisiae (PDB entry 3V8E) [10], Streptococcus mutans (PDB entry 3S2S) [21], and Acinetobacter baumannii (PDB entry 2WT9) [21], have previously been determined. These proteins all belong to the isochrismatase superfamily that also includes isochorismatase [22]. Despite the structural similarities between these two enzymes, nicotinamidase is characterized by the universal conservation of the Asp-Arg-Cys catalytic triad and its related chemistry, whereas isochorismatase is not [23,24]. Among these reported structures, the overall fold of PncA is strongly structurally similar to molecules composed of an α/β domain and a subdomain [13,20,21,25]. M. tuberculosis PncA is composed of a single domain that is nearly entirely made up of β-sheet structures [20]. Its structure also revealed the coordination of the iron ion in PncA within a conserved tetrad of amino acids consisting of one aspartate and three histidine residues, as well as two coordinated water molecules [20]. In contrast, PncA adopts a novel fold composed of two completely α-helical domains when complexed with its substrate [24]. Researchers have not yet determined whether unliganded or unsubstrated PncA has the same structure as the complexed form. Thus, further studies on the metal- and substrate-binding sites of this protein are required.
PncA has also been identified and investigated in Bacillus subtilis (B. subtilis), where it plays important roles in diverse biological processes [26]. However, the crystal structure of PncA from B. subtilis has not yet been published. Therefore, the precise mechanisms underlying its catalytic activity have yet to be completely elucidated. Thus, studies aiming to determine the three-dimensional structure of BsPncA are important. The functional and structural features of BsPncA provide a basis for the design and engineering of B. subtilis proteins that have specific and desirable functions.
We determined the crystal structure of BsPncA at a resolution of 2.0 Å to obtain a better understanding of the molecular basis of ligand recognition by BsPncA. Using this structure, we present the structural and functional characterization of PncA from B. subtilis that provides insights into its oligomeric state, ligand binding, and catalysis.
Section snippets
Protein preparation
The gene encoding BsPncA (residues 1–183) was PCR-amplified from B. subtilis genomic DNA. The resulting DNA fragments were then cloned into a pPROEX-HTA vector (Invitrogen, USA) between the NcoI and XhoI restriction sites, and the resulting pPROEXHTA-PncA plasmid contained a hexa-histidine tag at the N-terminus and a Tobacco etch virus (TEV) protease recognition site. Escherichia coli BL21 (DE3) cells were then transformed with pPROEXHTA-PncA, after which the transformants were grown in LB
Overall structure of BsPncA
We determined the crystal structure of B. subtilis PncA at 2.0 Å resolution to obtain a better understanding of its catalytic mechanism. The space group of the BsPncA crystals was P62 (hexagonal) with unit-cell parameters a = b = 90.04 Å and c = 86.18 Å. Two BsPncA molecules are present in the asymmetric unit and form a dimer (see below). The electron densities of all residues of BsPncA (Met1-Glu183) were well defined. The refined model was obtained with an Rwork of 17.35 and an Rfree of
Conflicts of interest
The authors have no conflicts of interest to report.
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 31200556 to Y. Xu and Grant No. 21272031 to C. Quan), the Program for Liaoning Excellent Talents in University (Grant No. LJQ2015030 to Y. Xu), the Fundamental Research Funds for the Central Universities (Grant No. DC201502020203 to Y. Xu, Grant No. DC201502020201 to C. Quan), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MOE) (Grant No. NRF-2017R1D1A1B03033087 and
Acknowledgements
The authors acknowledge the staff of the Pohang Light Source (PLS, Pohang, Republic of Korea) for their assistance with data collection.
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Both authors contributed equally to this work.