The activation of glycerol dehydrogenase by ppGpp Huyen Nga Hoang, Thanh Tuyen Tran, and Che-Hun Jung Department of Molecular Medicine, Department of Chemistry Graduate school, Chonnam National University, Gwangju, Korea 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, ppGpp binds to GldA and activates its 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. 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. The dissociation constants of NAD , NADH, and dihydroxyacetone, the substrate and products for GldA were 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, K . Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol. It is also suggested
that the + M 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. GldA - glycerol dehydrogenase TAB. 1. K for NAD , NADH, DHA and glycerol on GldA + D Molecular weight: 39535.05 Da Theoretical pI: 5.09 Glycerol + NAD ↔ Dihydroxyacetone + NADH + H + + GldA from Escherichia coli is a Zn -containing alcohol dehydrogenase which 2+ catalyzes the NAD -dependent oxidation of glycerol to
dihydroxyacetone. + FIG. 1. Determination of K for NAD on GldA by fluorescence spectrometry. + D (A) The fluorescence spectra of GldA when mixed with various concentrations of NAD . + (B) (B) The fluorescence changes (ΔF) at 320 nm against NAD concentrations. The K D + value for NAD was calculated as 110 ± 5 μM. + TAB. 2. The dissociation constants (K ) for D ppGpp, GTP and GDP on GldA FIG. 2. Activation of GldA by ppGpp. (A) A saturation curve against ppGpp concentration. (B) A double reciprocal plot of the % activation versus ppGpp concentrations. The half-maximal activation occurred at 33.1
± 3.1 µM. TABLE 3. The inhibition constants for Tris, GDP, and GTP on GldA CONCLUSION Glycerol dehydrogenase (GldA) from Escherichia coli is a FIG. 3. Determination of K for ppGpp on GldA by fluorescence spectrometry. Zn -containing alcohol dehydrogenase which catalyzes the D 2+ NAD -dependent oxidation of glycerol to dihydroxyacetone. + In this study, - ppGpp binds to GldA and activates its activity for the first time with the half maximal activation at 33.1 ± 3.1 µM. - GTP and GDP bind to GldA as strongly as ppGpp but only ppGpp activates GldA. - The dissociation constants of NAD , NADH, and
+ dihydroxyacetone, the substrate and products for GldA were 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 + FIG. 4. The ppGpp-binding site proposed by a molecular modeling study. kinetic constant, K M The simulation for ppGpp binding to GldA was performed by using GalaxyWEB and the generated model - Tris(hydroxymethyl)aminomethane serves as a competitive was visualized using PyMol. inhibitor against glycerol. - 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 ppGpp relatively lower affinities. ppGpp-binding site Active site: Asparagine 145, 148 Phe245, His254, Asp171, His271 Email: hoanghuyennga91@gmail.com First published:17 December 2019 https://doi.org/10.1002/bkcs.11932 [U. Protein structure and function-1] The activation of glycerol dehydrogenase by ppGpp Huyen Nga Hoang¹, Che-Hun Jung¹˙* ¹Molecular Medicine, Chonnam National University, Gwangju 61186, Korea Glycerol dehydrogenase (GldA) from Escherichia coli is a
Zn2+-containing alcohol dehydrogenase, catalyzing the NAD+-dependent oxidation of glycerol to dihydroxyacetone. Accumulation of ppGpp initiates stringent response of bacteria under nutritional stress. In this study, ppGpp binds to GldA and activates its activity with the half-maximal activation at 33.1 ± 3.1 µM. GTP and GDP also bind to GldA with similar affinity to ppGpp, however, they have no effect on GldA activity considering the physiological conditions. ppGpp activated GldA while Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol with the inhibition
constants (Ki) of 410 ± 21 µM. The interactions of E. coli GldA with its substrates and products were examined by both intrinsic fluorescence quenching and enzyme kinetics studies. The dissociation constants of NAD+, NADH, and dihydroxyacetone for GldA were 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. Our results suggest that the intrinsic fluorescence change of proteins by ligand binding is sufficiently sensitive for studying protein-ligand interactions. Binding of Glutathione and ppGpp to Stringent
Starvation Protein A (SspA) Taner Duysak and Che-Hun Jung Department of Molecular Medicine, Department of Chemistry Graduate School, Chonnam National University, Gwangju, Korea (Abstract) Stringent starvation protein A (SspA) is a glutathione S-transferase homolog. In this study, his6-tagged SspA from Escherichia coli has been cloned and over-expressed. SspA binds glutathione and 1-chloro-2,4-dinitrobenzene, the substrates for glutathione S-transferases, with the dissociation constants as 225.0 ± 34.4 μM and 75.3 ± 4.3 μM, respectively. This observation is contradictory to the previous report
that SspA, lacking glutathione S-transferase activity, does not bind glutathione. It has been reported that SspA is an RNA polymerase-associated transcription factor and that a functional relA gene is required for SspA to affect gene expression. A function of relA is to synthesize ppGpp, a global regulator in replication, transcription, and translation. This study shows for the first time that SspA binds ppGpp with the dissociation of constants of 109.1 ± 7.2 μM. This study may provide an insight why relA is required for regulating gene expression by SspA. Figure 4. Determination of the
dissociation constant (KD) for CDNB on SspA by Sequence alignment of E. coli SspA and GstA. fluorescence spectroscopy. (a) The fluorescence spectra of SspA in the presence of various concentrations of CDNB. While excited at 280 nm, the emission spectra of SspA SspA MAVAANKRSVMTLFS--GPTDIYSHQVRIVLAEKGVSFEIEHVE----KDNPPQDL (10 μg/mL) were recorded at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330 nm against M LF G + SH I L E G F + V+ + D CDNB concentrations. GstA ----------MKLFYKPGACSLASH---ITLRESGKDFTLVSVDLMKKRLENGDDY Binding of ppGpp to SspA SspA
IDLNPNQSVPTLVDRELTLW-ESRIIMEYLDERFPHPPLM-PVYPVARGESRLYMH +NP VP L+ + TL E IM+YL + P L+ PV ++R ++ +++ GstA FAVNPKGQVPALLLDDGTLLTEGVAIMQYLADSVPDRQLLAPVNSISRYKTIEWLN SspA seems not to be required for normal growth conditions for bacteria, and its synthesis is dramatically stimulated by a stringent response. It is also reported that a SspA RIEKDWYTLMNTIINGSASE--ADAARKQLREELLAIAPVFGQKPYFLSDEFSLVD functional relA gene is required for SspA to regulate gene expression in E. coli. Numerous I + + + E R QL ++L + + + F++ D studies suggested that the stringent response is characterized by the synthesis of
ppGpp, GstA YIATELHKGFTPLFRPDTPEEYKPTVRAQLEKKLQYVNEALKDEHWICGQRFTIAD which is catalyzed by relA. Up to date, the molecular basis how ppGpp is required for SspA CYLAPLL-WRLPQLGIEFSGPGAKELKGYMTRVFERDSFLASLTEAEREMRLGRS SspA function in gene expression is largely unknown. The fluorescence intensity of SspA YL +L W ++ + G + + +M R+ ER decreased in the presence of ppGpp (Figure 5), which allowed us to determine the GstA AYLFTVLRW---AYAVKLNLEGLEHIAAFMQRMAERPEVQDALSAEGLK------ dissociation constant. Figure 1. Forty seven amino acids are identical. The additional positive 35 amino acids are marked as
+. This figure shows that the amino acid sequence of SspA is not much homologous to GSTs, although their three-dimensional structures are well conserved. Figure 5. Determination of the dissociation constant (KD) for ppGpp on SspA by fluorescence spectroscopy. (a) The fluorescence spectra of SspA in the presence of various concentrations of ppGpp. SspA was excited at 280 nm and the emission spectra of SspA (10 μg/mL) were measured at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330 nm against ppGpp concentrations. Figure 2. Structural comparisons of E. coli GstA and SspA. (a) Crystal structure
of GstA. (b) The structure of SspA was generated by SWISS-MODEL(1yy7) and visualized by PyMol.25 Escherichia coli SspA is highly similar to GstA structurally. The Cys11 and Table 1. Dissociation constants KD of SspA for ppGpp, CDNB, GSH, and His106 at the active site of GstA are shown in (a) and the corresponding Tyr21 and glutathionesulfonic acid. Tyr111 are in (b). Three tryptophan residues, responsible for the intrinsic fluorescence of SspA are also visualized. KD (µM) Biding of GSH and CDNB to SspA Examined by Fluorescence ppGpp 109.1 ± 7.2 Spectroscopy. CDNB 75.3 ± 4.3 Escherichia coli
SspA showed a strong intrinsic fluorescence at 330 nm when excited at GSH 225.0 ± 34.4 280 nm. Three tryptophan residues in SspA may contribute to the fluorescence. The Glutathionesulfonate 272.0 ± 52.1 fluorescence intensity of SspA decreased in the presence of GSH, CDNB, and glutathionesulfonate (Figures 3 and 4). (Conclusion) SspA from E. coli shows a sequence homology to glutathione S- transferase and acts as a transcription factor. Although SspA does not have glutathione S-transferase activity, it binds strongly with both substrates, GSH and CDNB. SspA also binds with ppGpp, which may
provide the molecular basis why relA, the gene for ppGpp synthetase, is required for gene regulation by SspA. SspA may act like DksA, a ppGpp-binding transcription factor identified Figure 3. Determination of the dissociation constant (KD) for GSH on SspA by fluorescen previously. ce spectroscopy. (a) The fluorescence spectra of SspA in the presence of various concent rations of GSH. While excited at 280 nm, the emission spectra of SspA (10 μg/mL) were recorded at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330 nm against GSH concentrati ons. [U. Protein structure and function-2] Stringent
starvation protein A, a novel ppGpp-binding protein in E. coli TANER DUYSAK¹, Che-Hun Jung¹ ¹Molecular Medicine , Chonnam National University, Gwangju 61186, Korea Stringent starvation protein A (SspA) is an RNA polymerase-associated protein and its expression is induced by starvation for glucose, nitrogen, phosphate or amino acids or upon phage λ infection. SspA itself also serves as a transcriptional regulator. SspA shows a sequence homology to glutathione S-transferases and the crystal structure of SspA from Yersinia pestis reveals that SspA is structurally conserved to glutathione
S-transferases. It is reported, however, that SspA from various sources lacks glutathione S-transferase activity. The alarmone ppGpp, first reported by Cashel and Gallant, serves as a master regulator not only for the bacterial response to stress but also for almost all aspects of bacterial physiology, virulence, and immune evasion. In this study, SspA from Escherichia coli has been cloned and overexpressed. Here we report that ppGpp binds to SspA. On the contrary to the previous reports, SspA from Escherichia coli shows glutathione S-transferase activity. The binding affinity of ppGpp, GSH,
1-chloro-2,4- dinitrobenzene to SspA was determined by fluorescence spectrometry, and the KD
