Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum
Introduction
Mycobacterium marinum (M. marinum) is a saprophytic atypical mycobacterium, widely found in seawater and freshwater. The natural hosts of M. marinum are fish and amphibians [1]. M. marinum is a conditional pathogen, mainly resulted in skin infections [2]. The systemic granulomatous disease caused by Mycobacterium algae in its host is also similar in its main histological features to human tuberculosis, caused by the pathogenic Mycobacterium tuberculosis [3]. As the M. marinum genome shows high similarity (85%) with that of M. tuberculosis [4], rifampicin, ethambutol, and quinolone, as well as other drugs (such as doxycycline and clarithromycin), can be used to effectively treat M. marinum diseases [5,6]. The close relationship between the two Mycobacterium species is also demonstrated by the observation that a mutational knockout of virulence gene EspE/F in M. marinum is complemented by the ortholog gene of M. tuberculosis [7].
Sugar is the primary carbon source utilized by heterotrophic organisms, and its phosphorylation plays a key role in host cell metabolism [8,9]. The phosphorylation of sugars (mainly monosaccharides) is catalyzed by at least three different non-homologous protein enzyme families: the hexokinase family, the galactokinase family, and the ribokinase family [[10], [11], [12]]. Members of the ribokinase family catalyze the phosphorylation of many compounds, including ribose, fructose, nucleosides, and phosphorylated monosaccharides such as phosphate glucose [[13], [14], [15]]. Phosphofructokinase B (PfkB) belongs to the ribokinase family, which uses the phosphorylated sugar as substrate, and catalyzes the conversion of fructose-6-phosphate into fructose-1,6-diphosphate. The E. coli ribokinase structure in complex with D-ribose and a nucleotide analog was resolved at 1.84 Å resolution (PDB code: 1RKD) [16]. Afterward, the structure of Staphylococcus aureus 6-phosphoglucokinase was solved (PDB code: 2JGV) [17]. Most of the enzymes in this family are dimeric, which contained two domains, a large domain containing a three-layer sandwich structure of α/β/α, and a small domain consisting of four-strand β-sheets. The active site of the enzyme is in the crevice formed by the large and small domains; the sugar site is located near the hinge between the two domains, and the ATP site is located at the far end of the hinge. However, the structural basis of Mycobacterium marinum PfkB is not clear. In this work, the crystal structure of M. marinum PfkB at a resolution of 2.21 Å was solved by the molecular replacement method. Structural comparison and SAXS analysis showed that the two-domain protein PfkB exhibited a classical fructokinase structure. This implies that the homology to other kinase structures with this fold supported the assignment of this enzyme as acting in the glycolytic process in Mycobacterium.
Section snippets
Protein expression and purification
The genes encoding PfkB (WP_020729646.1) of Mycobacterium marinum was synthesized and constructed into the pSMT3 vector to produce the N-terminal 6xHis-SUMO tagged fusion protein. PfkB was expressed in Escherichia coli BL21(DE3) cells. Cell cultures in LB medium were induced with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) at 18 °C for 20 h when OD600 reached 0.6–0.8. Then, cells were suspended in buffer A (20 mM Tris-HCl, pH8.0, 500 mM NaCl, 5% glycerol), and lysed by a high-pressure
Expression, purification, and enzymatic characterization of the PfkB
PfkB from Mycobacterium marinum was synthesized and constructed into the pSMT3 vector. Then, PfkB was expressed in Escherichia coli (DE3) cells and purified as previously mentioned. The high purity PfkB came out at a peak of ∼87 mL on the Superdex200 16/600 column, which was corresponding with molecular weight 34 kD. Therefore, PfkB was a monomer in solution, as the theoretical molecular weight of PfkB is 33 kD (Fig. 1A and B). The kinase activity of PfkB was assayed with a spectrophotometric
Discussion
M. marinum maintains a huge genome to preserve environmental viability and, at the same time, becomes a similar pathogen to M. tuberculosis with a wide range of hosts. Genome sequencing revealed that M. marinum has complete glycolysis and pentose phosphate pathways. PfkB, which belonged to the ribokinase (RK) family, was one key enzyme in the glycolysis process. Most reported ribokinase and PfkB proteins function as dimers and the interactions between monomers occur mainly across the lid
Author contributions
J.L. conceived and supervised the study. B.G., R.J., X.S., Z.L., H.L., Y.S., C.J., and J.G. performed experiments and data analysis. J.L., and B.G. wrote the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank the staff from the BL17U1 beamline at Shanghai Synchrotron Radiation Facility (SSRF) and BL18U1/BL19U1/BL19U2 beamlines of the National Center for Protein Sciences Shanghai (NCPSS) for their assistance in the data collection process. This work was funded by grants from the Nation Key Research and Development Program of China(2016YFA0500600), the Nation Natural Science Foundation of China (31670878), and the Shanghai Committee of Science and Technology (18430711400).
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These authors contributed equally to this study.