Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis
Introduction
The etiological agent of tuberculosis, Mycobacterium tuberculosis (Mtb), has infected nearly one-third of the human population [1]. Approximately 10% of TB-infections lead to an active, symptomatic infection that resulted in nearly 1.4 million deaths in 2011 [1]. In addition, multi-drug resistant strains have been reported in every country surveyed by the World Health Organization [1]. Yet some of the most basic metabolic enzymes of this bacterium have yet to be characterized.
Glyceraldehyde 3-phosphate dehydrogenase is a highly conserved enzyme that is utilized in central carbon metabolism by some of the most ancient forms of life [2]. GAPDH is best known for its role in glycolysis, catalyzing the reversible conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and NAD+ to 1,3-bisphosphoglycerate (1,3-BPG)1 and NADH [3]. This dehydrogenase is also unusual in that it utilizes a covalent thiohemiacetal intermediate to promote hydride transfer and catalysis [3]. The reaction of GAPDH is essential for the regeneration of the two molecules of ATP used to phosphorylate the hexose carbon source, glucose. The cleavage of fructose-1,6-bisphosphate yields the two triose phosphates that are interconverted into G3P. The oxidation of the aldehyde and substrate-level phosphorylation catalyzed by GAPDH generate NADH and the high energy carboxy-phosphoric anhydride containing 1,3-bisphosphoglycerate (1,3-BPG) that is used in the subsequent reaction catalyzed by 3-phosphoglycerate kinase to regenerate the two molecules of ATP used earlier in the glycolytic sequence. The very reactive nature of the product of GAPDH, 1,3-BPG, has recently been shown to be capable of non-enzymatic modification of proteins, including GAPDH [4].
Recent studies have also found GAPDH to be involved in a variety of cellular processes in addition to its major role in glycolysis. GAPDH has been shown to play a role in transcription, assisting in the formation of both DNA and RNA binding complexes as well as acting as a transcription factor co-activator [5], [6], [7]. Additionally, GAPDH has been identified as a microtubule-binding protein, a lactoferrin receptor, and as an apoptosis-inducer [8], [9], [10], [11]. More information on the extra-glycolytic roles of GAPDH can be found in the review by Nichollis et al. [12].
Despite decades of work on GAPDH’s from prokaryotic and eukaryotic sources, no work has been conducted on the GAPDH from M. tuberculosis. It was discovered early on in our work that this enzyme had significant solubility issues. This obstacle was overcome by co-transforming the Mtb-GAPDH plasmid along with a plasmid expressing the chaperones GroEL/GroES [13]. These chaperones are believed to create an environment suitable for proper folding yielding soluble and active Mtb-GAPDH [13]. In this study, we report the first successful purification and mechanistic evaluation of Mtb-GAPDH using steady-state kinetics, pH-rate profiles, isotope effects and mutagenesis to elucidate both the kinetic and chemical mechanism of Mtb-GAPDH.
Section snippets
Materials
All chemicals were purchased from Sigma–Aldrich unless otherwise noted. The Mtb-GAPDH gene was cloned into the Novagen pET-28a(+) vector. The GroEL/GroES plasmid was a gift from the Shrader Lab [13]. Primers were purchased from Invitrogen. All cloning enzymes and T7 competent Escherichia coli were purchased from New England Biolabs. Complete EDTA-free protease inhibitor cocktail and DNase were purchased from Roche. 99.9% deuterated water was purchased from Cambridge Isotope Laboratories.
Cloning, expression and purification of Mtb-GAPDH
The M.
Cloning, expression and purification
Mtb-GAPDH was PCR amplified then cloned into the pET28a expression vector encoding a N-terminal His6-tagged Mtb-GAPDH. Sequencing confirmed that no mutations were introduced during the cloning process. Initial expression studies revealed that Mtb-GAPDH was insoluble under normal conditions. Co-transformation of the Mtb-GAPDH plasmid along with a GroEL/GroES plasmid yielded soluble and active protein. After purification and dialysis, the final protein preparation was >95% pure as determined by
Chemical mechanism
The chemical mechanism for Mtb-GAPDH shown in Scheme 3 is supported by our determination of the kinetic mechanism, mutagenesis and isotope effect studies. The “free enzyme” is actually present with bound NAD+, and the two important catalytic residues, Cys158 and His185, are present as a thiolate-imidazolium ion pair. Binding of glyceraldehyde-3-phosphate yields an initial Michaelis complex, and the Cys158 thiolate nucleophilicially attacks the aldehyde to generate the neutral thiohemiacetal
Acknowledgments
We thank Drs. Subray Hegde and Hector Serrano for the assistance in the purification of Mtb-GAPDH. This work was supported by the National Institutes of Health Grant A1060899 to J.S.B.
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2019, Metabolic EngineeringCitation Excerpt :In the analysis we have presented here, we decided not to adjust for 2H-KIEs in flux calculations for the following reasons (Shui et al., 2015): the experimental approach that we employed in this study was designed to minimize the effects of potential 2H-KIEs on flux calculations by integrating 2H and 13C tracers into a single MFA model. This approach avoids reliance on isolated 2H-tracer data and provides redundancy and cross-validation of results (Gu et al., 2015); the reported in vitro 2H-KIEs associated with GAPDH and ENO, the reversible ED pathway reactions potentially affected by KIEs when using, respectively, [4-2H] and [5-2H]glucose, are only modest; varying between 1.2 and 1.9 for GAPDH and 1.2–2.1 for ENO in different organisms (Liu and Huskey, 1992; Wolfson-Stofko et al., 2013; Canellas and Cleland, 1991; Shen and Westhead, 1973; Anderson et al., 1994). In addition to these two arguments, adjusting for KIE would require making assumptions about how KIE measured in vitro may affect metabolic flux in vivo, potentially introducing additional uncertainty to the model instead of increasing its precision, particularly when the KIE is only modest (Millard et al., 2015).
Expression of glyceraldehyde-3-phosphate dehydrogenase from M. tuberculosis in E. coli. Purification and characteristics of the untagged recombinant enzyme
2019, Protein Expression and PurificationCitation Excerpt :If some amount of the recombinant protein is present in the extract, it exhibits no enzymatic activity. Previously, it was reported that the isolation of His-tagged M. tuberculosis GAPDH was possible only in the case of the co-expression of the protein with the chaperonin GroEL/GroES [14]. We assumed that the production of inactive and insoluble Mtb-GAPDH_His could be due to the presence of the additional construction on its N-terminus that hindered the folding of the recombinant protein, and the removal of this construction might help in the production of soluble and active enzyme.
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