The Activation of Glycerol Dehydrogenase from Escherichia coli by ppGpp

Abstract

Glycerol dehydrogenase (GldA) from Escherichia coli is a Zn2+‐containing alcohol dehydrogenase which catalyzes the NAD+‐dependent oxidation of glycerol to dihydroxyacetone. In this study, GldA has been cloned, over‐expressed, and isolated by an affinity and an ion‐exchange chromatography. GldA shows a strong intrinsic fluorescence at 320 nm, when excited at 280 nm. The fluorescence intensity decreases in the presence of NAD+, NADH, and dihydroxyacetone, the substrate and products for GldA, which allows us to determine the dissociation constants for those molecules as 110.6 ± 5.0 μM, 9,1 ± 0.6 μM, 33.3 ± 2.3 mM, respectively. The dissociation constant for NAD+ was similar to the kinetic constant, KM. Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp), accumulated in E. coli when starved for amino acids, nutrients, and phosphate, serves as a global regulator in replication, transcription, and translation. In this study, the fluorescence intensity of GldA also decreases in the presence of ppGpp and the dissociation constant for ppGpp is calculated as 108.9 ± 8.6 μM. ppGpp increases GldA activity with the half maximal activation at 33.1 ± 3.1 μM. On the contrary, GTP and GDP inhibit GldA, with the inhibition constants of 16.1 ± 1.1 mM and 10.6 ± 0.3 mM, respectively. Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol. GTP and GDP also bind to GldA with the dissociation constants of 60.0 ± 0.8 and 61.0 ± 1.3 μM, respectively. These results suggest that GTP and GDP bind to GldA as strongly as ppGpp but only ppGpp activates GldA. This study shows that ppGpp binds to GldA and activates its activity for the first time. It is also suggested that the strong intrinsic fluorescence of enzymes and their changes in the presence of various ligands can be utilized to measure the binding affinities for those ligands. The method described here is especially effective for bindings with the relatively lower affinities.

Structural and functional insights into the flexible β‐hairpin of glycerol dehydrogenase

“…Our structures thus show that Tris was bound at the central cleft, including the active site, where it coordinated with the catalytic Zn 2+ as the substrate of GDH does and that they formed a ternary complex with NAD + . The catalytic Zn 2+ coordination of Tris is consistent with the earlier finding that Tris inhibits GDH by competing with glycerol [13]. In addition, a homooctameric assembly of GDH NAD•Tris was also formed by the crystallographic symmetry operations, and the two protomers of GDH NAD•Tris structure had nearly identical conformations, not only for their overall structures, which had a RMSD of 0.4 Å for 367 C α atoms, but also for the β-hairpin.…”

“…Nonetheless, in all the ASU molecules of our two structures, there were additional electron densities adjacent to the catalytic Zn 2+ , where water molecules were insufficient to fill. Given the crystallization conditions and references showing that Tris can be located at the active site of GDH [13,16], we fitted Tris to the additional electron density maps observed at the active site. To prove the existence of Tris and optimize its positions for the best fit, we utilized calculation of its omit F o À F c map with simulated annealing that minimizes model bias, occupancy refinement for each Tris and dual conformational fitting when necessary.…”

“…In this regard, it is unclear why and how GDH strictly requires the binding of NAD + prior to the substrates. Given that GDH has a relatively high K m for glycerol (an average of ∼ 50 mM) [13,20,[22][23][24][25] and that glycerol and its analogues can also bind at various sites along the central cleft [17], it appears that glycerol may move along the cleft rather than being specifically located only at the active site. If so, prior binding of glycerol could potentially hinder NAD + 's access to the peripheral area of the active site, along this cleft.…”

“…Consequently, metal-chelating agents such as 8hydroxyquinoline act as inhibitors of GDH [11]. Competitive inhibition by substrate analogues has also been investigated, and tris(hydroxymethyl)aminomethane (Tris) has been shown to inhibit GDH with an IC 50 of 2 mM [12,13]. Biochemical studies have also revealed that GDH is able to utilize a broad range of diols, including 3-mercapto-1,2-propanediol, 1,2-propanediol, 2,3-butanediol and 3-methoxy-1,2-propanediol, as its substrates [8,10,11].…”

Binding of Glutathione and ppGpp to Stringent Starvation Protein A (SspA)

“…ppGpp controls bacterial replication, transcription, and translation 12 . In addition, ppGpp directly regulates many enzymes, such as glycerol dehydrogenase, polyphosphate kinase, lysine decarboxylase, DNA primase, and translation factors 13,14 . It has been reported that the regulation of gene expression by SspA requires a functional relA gene, 2 which is responsible for synthesizing ppGpp from GTP/GDP and ATP 15,16 .…”