Enzymatic Characterization of Fructose 1,6-Bisphosphatase II from Francisella tularensis, an Essential Enzyme for Pathogenesis
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The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis. We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: Km 11 μM, Vmax 2.0 units/mg, kcat 1.2 s−1, kcat/Km 120 mM−1 s−1, and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme’s properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis.
KeywordsGluconeogenesis Francisella tularensis Fructose-1,6-bisphosphatase Enzymatic activity glpX
Class I Fructose-1,6-bisphosphatase gene
Class II Fructose-1,6-bisphosphatase protein
Fructose-1,6-bisphosphatase from Francisella tularensis
Class II Fructose-1,6-bisphosphatase gene
E. coli BL21(DE3) strain lacking fbp gene
JB108 transformed with empty plasmid
JB108 transformed with plasmid containing glpX gene
E. coli strain lacking fbp gene and glpX gene
E. coli strain lacking fbp gene
Size exclusion chromatography
Francisella tularensis (F. tularensis) is the etiologic agent of tularemia. This highly virulent bacterium is considered high risk for use as a biological weapon since the minimal infectious dose for human is only 10 bacteria . Identifying new drugs and antibiotics against such pathogens is vital. Therefore, the execution of a rational structure-based drug design study targeting key regulatory proteins and enzymes in F. tularensis would ultimately lead to a novel antibiotic which could potentially become a drug.
The virulence of several F. tularensis SCHU S4 strain mutants was assessed by following the outcome of infection after intradermal infection. In this study, the virulence of 20 in-frame deletion mutants and 37 transposon mutants were assessed . The majority of the mutants did not show an increase in prolonged time to death. However, mutations in six unique targets resulted in significantly prolonged time to death and mutations in nine targets, including glpX, and led to marked attenuation with an LD50 of >103 CFU. Compared to the wild-type strain with the LD50 of one CFU, glpX mutant showed a marked attenuation with an LD50 of 107 CFU or greater . The extreme attenuation of the ∆glpX mutant suggests that glpX is required for the virulence of F. tularensis in vivo .
More recently, the glpX of F. tularensis was demonstrated to be an essential component for bacterial growth and a requirement for mouse infection . Growth of wild-type and the ∆glpX mutant of F. tularensis were unaffected by glucose and ribose, while the ∆glpX mutant greatly impaired growth on glycerol, pyruvate, and an amino acid cocktail. A challenge of wild-type and ∆glpX mutant F. tularensis, subspecies F. novicida, in mice resulted in an 80% survival rate for mice infected with the ∆glpX mutant after 10 days, while mice who received the wild-type bacteria all died after 3 days .
Our work on the glpX encoded FBPase II in Mycobacterium tuberculosis indicates that it is essential for the long-term survival and proliferation in mice model . We were able to successfully demonstrate that the glpX encoded FBPase is an FBPase II . Further, we reported the expression, purification and biochemical characterization of this enzyme in M. tuberculosis [6, 7]. The sequence similarity of the glpX-encoded proteins in both M. tuberculosis and F. tularensis promoted us to investigate the properties of this protein in F. tularensis as well.
There are five known classes of FBPases [8, 9, 10]. Most organisms contain a mixture of Class I and II. Class I FBPases, the most common form, are found in several eukaryotes and some prokaryotes while Class II is mainly found in bacteria with a few cases in eukaryotes . Class III was defined for a unique FPBase from Bacillus substilus, structurally unrelated to the other classes . Class IV from archaea have both inositol monophosphatase and fructose-1,6-bisphosphatase activity . Class V FBPases are found in thermophile prokaryotes and contain an unusual 4-layer α-β-β-α fold instead of the common 5-layer α-β-α-β-α fold . Escherichia coli has both Class I (fbp) and Class II (glpX). To date, F. tularensis contains one proposed FBPase Class II, glpX. The glpX gene is required for the virulence of F. tularensis in vivo [2, 3]. There is no known Class I fructose 1,6-bisphosphatase (FBPase) in F. tularensis, and it is expected that the glpX gene product is responsible for the catalysis of fructose 1,6-bisphosphate (F16BP) due to sequence similarity. However, the exact biochemical activity, or function of the glpX gene product, fructose 1,6-bisphosphatase (FBPaseII), has not been experimentally verified. Here, we have biochemically characterized the FBPaseII of F. tularensis (FtFBPaseII) to verify functionality, specificity and stability. We also describe preliminary structural characterization by size exclusion chromatography of FtFBPaseII. These experiments serve as a foundation for a structure-based drug design strategy targeting the FtFBPaseII enzyme.
Materials and Methods
All materials were purchased from Thermo Fisher Scientific, Waltham, MA, unless noted. Enzymes for the secondary assay and substrate compounds were purchased from Sigma Aldrich, St. Louis, MO.
The F. tularensis glpX gene was codon optimized, synthesized, and cloned into a pET15b vector suitable for genetic complementation studies and recombinant expression (CelTek Bioscience, Franklin, TN). The construct was sequenced for its accuracy, subjected to restriction digestion with BamHI/XbaI, and the insertion of the FtglpX gene was confirmed.
The pET15b-FtglpX construct was transformed into E. coli strain JLD2402 (TL524 glpX::Spcr Δfbp287 zjg920::Tn10), which lacks both fbp and glpX, (JLD2402-pET15b-FtglpX), and JLD2404 (TL524 glpX + Δfbp287 zjg920::Tn10), which lacks only fbp (JLD2404-pET15b-FtglpX) . Antibiotic-resistant transformants were grown on minimal media plates containing glucose or glycerol as the sole carbon source and isopropyl β-d-1-thioglactopyranoside (IPTG) to induce expression of the glpX gene. Additionally, the pET15b-FtglpX construct was transformed into E. coli strain JB108 (BL21(DE3) zjg920::Tn10 ∆fbp287) (JB108::pET15b-FtglpX) , which was grown in minimal medium containing glycerol. Appropriate control strains of E. coli BL21(DE3) (Novagen, Billerica, MA), untransformed JB108, and JB108 strain transformed with pET15b (JB108::pET15b) were also run with appropriate concentrations of IPTG. The plates were incubated at 27 °C for 36 h to visually confirm bacterial growth.
Species Primary Sequence Comparison
The glpX sequence of F. tularensis was obtained from the KEGG database (FTF1631c), the E. coli sequence was obtained from SWISSPROT (accession no. P28860), the C. glutamicum sequence was obtained from GenBank (accession no. 19552240), the M. tuberculosis sequence was obtained from the Tuberculist server (http://genolist.pasteur.fr/TubercuList/; gene name Rv1099c), and the M. smegmatis sequence was derived from genomic sequences obtained from the Institute for Genomic Research website (http://www.tigr.org/). The exact start residue of the proteins is known only for C. glutamicum .
Expression and Purification
The pET-15b-FtglpX construct expressing N-terminal histidine-tagged FtFBPaseII was transformed into E. coli strain BL21(DE3). The positive colonies were grown on LB agar containing ampicillin (100 μg/mL). Then, the transformants were grown overnight in LB broth (with ampicillin at 100 μg/mL) at 37 °C in an incubator shaking at 180 rpm. After 16 h, 1.0 mL of this culture was transferred to 100 mL of LB broth (with ampicillin at 100 μg/mL) and incubated at 37 °C. When the OD600 reached 0.6, the culture was induced with IPTG (1 mM). After 5 h, the cell pellet was harvested and frozen at −20 or −80 °C until further use.
Purification of the FtFBPaseII was performed in a similar manner as described for MtFBPaseII , except that the final buffer exchange and concentration of the purified protein was performed using an Amicon-15 Ultracel 30 K centrifuge concentrator or Zeba spin desalting column. Protein molecular mass was determined using electron spray ionization mass spectrometry coupled with HPLC (Thermo Orbitrap Velos Pro MS, Agilent 1200 nano HPLC) through the Research Resources Center at UIC.
Size Exclusion Chromatography
Size exclusion chromatography (SEC) was performed on an ÄKTA purifier FPLC system in a similar manner to that described for MtFBPase . Retention times were determined by monitoring the absorbance at 280 nm. FtFBPaseII was injected at a concentration of 1.0 mg/mL. Total protein quantification was performed using the Pierce 660-nm kit. Bovine serum albumin (BSA) was used as the standard [ThermoFisher Scientific]. The relative elution (K av ) and hence the molecular weight for F. tularensis FBPaseII was estimated as described for MtFBPaseII using thyroglobulin (bovine) 670 kDa, gamma globulin (bovine) 158 kDa, ovalbumin (chicken) 44 kDa, myoglobulin (horse) 17 kDa, and vitamin B12 1350 Da. Since raw data was no longer available, previously acquired images were uploaded to http://arohatgi.info/WebPlotDigitizer and the data was processed with the Δx step interpolation algorithm of 0.01 units and 0% smoothing to create Fig. 4a, b (WebPlotDigitizer, Austin, TX).
Assays were conducted in 50 mM Tris pH 8.0 with 100 mM Mn2+, 50 nM FtFBPaseII, and 100 μM F16BP unless otherwise indicated. Protein concentration was determined by absorbance at 280 nm with extinction coefficient 12,950 M−1 cm−1. Assays were performed at room temperature, approximately 22–24 °C.
A malachite green assay  was used to test for metal requirements, buffer system, and substrate specificity. Enzymatic solutions were allowed to react for 15 min before being quenched with the malachite green agent. The absorbance at 630 nM was measured after an additional 5 min for dye development. The following metals, as chloride salts at 100 mM, were tested for absolute requirement for activity: Mg2+, Ca2+, Zn2+, Fe2+, Cu2+, Co2+, Ni2+, K+, Na+, and Mn2+. Different buffering systems were tested at pH 7.5 and 50 mM buffer component. The pH of 50 mM Tris was screened (7.0–9.0 pH units). The amount of phosphate released was determined by using a calibration curve of potassium phosphate standard in the presence of 50 mM Mn2+. Different substrates at 1 mM (d-fructose-1,6-bisphosphate, Sn-glycerol-6-phosphate, 3-phosphoglycerate, d-mannose-6-phosphate, d-fructose-6-phosphate, d-glucose-6-phosphate, d-fructose-1-phosphate, d-ribulose-1,5-bisphosphate, d-glucose-1,6-bisphosphate) were tested for substrate specificity against FtFBPaseII and adjusted for autophosphohydrolysis.
A coupled assay [6, 15], measuring production of NADPH at 340 nm, was used to determine enzymatic parameters and analyzed with a non-linear fit of Michaelis-Menten and kcat equations (fixing Et at 50 nM) using GraphPad Prism version 7.0b for Mac (GraphPad Software, La Jolla, CA). Using the coupled assay, the following optimal conditions were found where FtFBPaseII is the limiting reagent: 5 units/mL of phosphoglucoisomerase, 2 units/mL of glucose-6-phosphate dehydrogenase, 0.3 mM NADP+, 15 μM F16BP, and 50 nM FBPaseII. Li+ sensitivity was assessed with concentrations up to 100 mM. The reaction was monitored for 3.5 min. Enzyme inhibition by adenosine diphosphate (0.6–4 mM) and free phosphate (0.2–10 mM) was calculated relative to the unaccompanied enzyme. The residual activity after heating was assessed by incubating protein samples for 30 min at various temperatures in a water bath (10–80 °C), returning to ice for 15 min and assaying against a sample on ice for the same length of time.
F. tularensis glpX Encodes a Class II FBPase which Functionally Complements an E. coli ∆glpX/∆fbp Strain
In case of E. coli, it has been proven that the glpX-encoded FBPase II is not crucial for cell growth and proliferation since a strain lacking the glpX gene (JLD2403 (fbp1 glpX::Spcr)), successfully grows on LB or glucose, fructose, succinate, or glycerol minimal medium, in both aerobic and anaerobic conditions when the fbp-encoded FBPase I (major FBPase in E. coli) is present.
FBPase activity in E. coli strains expressing FtFBPaseII protein
FBPase specific activity (nmol min−1 mg−1)
16.30 ± 1.22
0.33 ± 0.08
0.09 ± 0.03
0.16 ± 0.07
0.33 ± 0.09
5.53 ± 1.13
9.89 ± 1.75
16.72 ± 1.25
21.46 ± 1.87
Molecular Weight Determination and Hydrodynamic Size
Mass spectrometry experiments indicated a mass of 36,846.6 g/mol, corresponding to the predicted size of 36,846 g/mol with the initial methionine cleaved in situ . The oligomeric state of the FtFBPaseII in solution state was evaluated by SEC. The void volume of the column, as determined by dextran blue, is V o = 39.80 mL; total column volume, V t (also referred to as geometric column volume) = 120 mL . Using this method, two protein peaks corresponding to molecular weights of 63.8 and 124.9 kDa were observed (Fig. 4b). This observation suggests that at a concentration of 1 mg/mL, F. tularensis FBPaseII exists as a mixture of both dimers and tetramers, assuming the monomer subunit is about 35 kDa.
Using a coupled assay with real-time measurements, Km was found to be 11 μM, Vmax was 2.0 units/mg, kcat was 1.2 s−1, kcat/Km was 120 mM−1 s−1, and specific activity of the enzyme was 2.0 units/mg. The R2 coefficient of determination was greater than 0.99. The reaction was linear for enzyme concentration up to 50 nM. FtFBPaseII was inhibited by Li+ with an IC50 value of 100 mM in the coupled assay. Adenosine diphosphate had 28% inhibitory activity of FtFBPaseII at 1 mM and precipitated at higher concentrations. High concentrations of phosphate also precipitated in the enzyme solution; nevertheless, 69% inhibition of the enzyme was detected at 150 μM.
Recent experiments have suggested the essentiality of the glpX gene in F. tularensis . These studies followed the growth of F. novicida, a subspecies of F. tularensis, in glucose and glycerol. While these studies demonstrate that the glpX-encoded protein is required for growth on gluconeogenic substrates, they do not functionally verify the enzymatic activity of the encoded protein.
Our experimental plan to verify the functional activity was based on complementation of known E. coli mutants lacking significant FBPase activity by the FtglpX-encoded protein. While the complementation with E. coli strains JLD2402 and JLD2404 does not rule out non-specific complementation, it does indicate that the FtglpX-encoded protein helps such strains grow on gluconeogenic substrates.
Complementation studies show that the pET15b-glpX plasmid was able to restore growth of the E. coli strains (lacking a functional FBPase I) on glycerol and independently verify that the glpX-encoded FBPase of F. tularensis is a functional FBPase. Since the E. coli control strains do not have FBPase enzymes, it can be interpreted that FBPase activity is needed for growth on glycerol. This work independently verifies that the glpX-gene encoded protein in F. tularensis is indeed an FBPase which can successfully complement a Δfbp E. coli strain (JB 108). Furthermore, the IPTG-induced overexpression of FBPase II from F. tularensis in a Δfbp E. coli strain (JB 108) by pET15b-FtglpX proves that overexpression of this protein allows growth and proliferation of the E. coli strain.
Additionally, the primary sequence comparison of FtFBPaseII with other known Class II FBPases also indicates that several regions are conserved and important catalytic residues in the enzyme are conserved. This work together with the complementation studies verifies that the encoded protein is an FBPaseII.
FBPase enzymes have been the subject of many drug discovery programs and other fields of research culminating with over 162 structures available in the protein data bank. A few of these enzymes have been subjected to extensive biochemical characterization, most notably, those from E. coli [14, 19], Corynebacterium glutamicum , and M. tuberculosis .
The E. coli FBPase is a dimer [14, 19] in solution while those from C. glutamicum  and M. tuberculosis  are both tetramers. Interestingly, we found that the quaternary structure was dependent on the protein concentration. While it is possible that there were two different molecular weight structures found at lower protein concentration, it is also possible that the protein had adopted two different conformations [20, 21]. In either case, we can conclude that the protein is more stable at the higher concentration.
FtFBPaseII is dependent on Mn2+ for activity, as is the E. coli enzyme  while those from C. glutamicum and M. tuberculosis can use either Mn2+ or Mg2+ . FBPase from M. jannaschii could substitute Zn2+ . F. tularenesis was found to have weak sensitivity to Li+, similar to E. coli with an IC50 of 70 mM . Li+ sensitivity in other organisms was more pronounced with IC50 values of 200 μM (M. tuberculosis)  and 140 μM (C. glutamicum) .
Substrate affinity and specificity between differing species’ enzymes may give us critical insight into active site differences. The Km for F. tularensis is comparable to C. glutamicum with a Km of 14 μM , while E. coli and M. tuberculosis Km values are higher at 35 μM  and 44 μM , respectively. Vmax of C. glutamicum of 5.4 units/mg  was highest, with E. coli Class II at 3.3 units/mg  and M. tuberculosis at 1.6 units/mg . kcat values of 1.0  M. tuberculosis and 3.2  C. glutamicum and 14.6  E. coli Class I s−1. While kcat/Km values had a larger range of 22.7  (M. tuberculosis), 57  (E. coli Class II), 948  (E. coli Class I), and 236 mM−1 s−1  (C. glutamicum). E. coli had low activity with substrates fructose l-phosphate and ribulose 1,5-bisphosphate  and glucose 1,6-bisphosphate . C. glutamicum has low activity with glucose-6-phosphate .
FBPases are known to require bivalent metal ions and have sensitivity to lithium. These differences in absolute metal requirement, substrate affinity, substrate specificity, and lithium sensitivity may be the key indicators for targeting a particular enzyme for structure-based drug design . The subtle active site changes from one species to another elucidated by structural analysis will give us critical insight. Substrate specificity differences give important enzymatic activity information for rational drug design . With the aid of computational energy minimization, examination of the binding differences of the tested substrates could be used as a starting point.
It is assumed that inhibition of the enzyme by phosphate is due to binding of phosphate in the active site where the substrate’s own phosphate groups would bind. However, inhibition by adenosine diphosphate (ADP) brings up questions of the presence of an allosteric site. AMP is an allosteric inhibitor of human FBPase . Future work to find the X-ray structure and further biochemical characterization with FtFBPase and ADP should tell us the mechanism of inhibition.
The genetic complementation results prove that the F. tularensis glpX gene encodes for a protein that possesses FBPase activity and can complement the E. coli ∆fbp strain. Bioinformatics results indicate that it is a Class II FBPase. FtFBPaseII was easily purified following standardized protocols. Biochemical characterization has provided valuable and novel information for drug discovery and should be pursued as an ongoing research activity.
The major bottlenecks in the process of structure-based drug discovery against this target are the availability of a purified protein target and the ability to crystallize the target in a robust crystal form. We have succeeded in the purification and biochemical characterization of the enzyme. The biochemical and structural understanding of this validated enzyme target can serve as a starting point for a structure-based drug discovery approach for this highly virulent bacterium.
We would like to acknowledge Professor Michael E. Johnson at the University of Illinois at Chicago for the use of the FPLC instrument and Professor Chuan He at the University of Chicago for providing help with the SEC.
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