Page 5 - U. Protein structure and function
P. 5
Malate Dehydrogenase a novel ppGpp binding protein
Rana Aqeel Afzal and Che-Hun Jung
Department of Molecular Medicine, Department of Chemistry
Graduate School Chonnam National University, Gwangju, Korea
Abstract
Titration with ppGpp for Kd determination
The citric acid cycle is the central oxidative pathway in prokaryotes and eukaryot (F) (G)
es under aerobic conditions. One of the reactions in the cycle is the conversion of
malate to oxaloacetate, catalyzed by NAD - or NADP -dependent malate dehydro
+
+
genase (MDH). Although MDH has been described as an enzyme for the oxidative
TCA cycle, the reaction that forms oxaloacetate is highly unfavorable thermodyna
mically. For this reason, it has been suggested that this enzyme has other functions
inside the cell. MDH from Escherichia coli under anaerobic conditions participate
s in a reductive TCA cycle (converting oxaloacetate to malate) that produces succi
nic acid. The alarmone ppGpp is involved in growth regulation and various stress
responses in bacteria. It induces profound transcriptional alterations, including th
e repression of stable RNA synthesis and induction of stress response factors and g
enes required for amino acid biosynthesis and transport. In this study, we report
MDH as a novel ppGpp-binding protein with a high affinity. No activity change as
sociated with the ppGpp-binding suggests that ppGpp does not bind at active site
of MDH. Further experiments for ppGpp-binding site on MDH, and its unidentifi
ed functions in E.coli are in process.
Cloning and Purification 1. MDH (10µM) prepared in buffer (50mM Tris, pH 7.5) and titrated 30 times with
10µl of 1.74mM ppGpp using fluorescence spectrophotometer F-4500 (Figure F).
2. The Kd value is determined by plotting graph between Delta F and ppGpp
Expression concentrations using Origin 9.1 software (Figure G).
Grew overnight mini-culture at 37˚C than transferred to one litter L 3. Calculated Kd value is 46.25 µM ± 2.80 µM at 295 nm.
B medium supplemented with ampicillin at a ratio of 1:100 and furth
er grown at 37˚C until the culture density (OD600) reached approxim (H) (I)
ately 0.6 and than put at 18˚C for 30min. Isopropyl-β -D-thiogalactop
yranoside (IPTG; Sigma) was then added to the cell culture to a final
concentration of 0.1 mM, and the cells were further incubated at 37˚
C for 4 hr.
Cell Lysis
The cells were harvested by centrifugation at 4000 Xg for 25 min at 4
˚C, and resuspended in ice-cold buffer consisting of 50 mM Tris, pH
8.0 and cells were disrupted by sonication.
Centrifugation
Cell debris was removed by centrifugation at 10,000 g for 30min at 4˚
C.
Affinity Chromatography 2cmd monomer front view 2cmd dimer front view
Crude cell supernatant applied to His Trap TM Ni HP(GE Healthcar
e) and run chromatography using buffers, (J) (K)
Buffer A (washing): 50 mM Tris-HCl, 10mM Imidazole, pH 8.0
Buffer B (Elution): 50 mM Tris-HCl, 500mM Imidazole, pH 8.0
Ion Exchange Chromatography
Eluent from affinity chromatography applied to His Trap TM Q HP
for removing impurities.
MDH activity with and without ppGpp
(B) (C) (A)
Predicted model for ppGpp bound to MDH using docking software
MDH
Summary
• MDH catalyzes the reversible oxidation of malate to oxaloacetate in TCA cycle.
Determination of Km values of OAA and NADH
(D) 4 (E) 4
• Although MDH has been described as an enzyme for the oxidative TCA cycle, the
reaction that forms oxaloacetate is highly unfavorable thermodynamically. For
3 3
this reason, it has been suggested that this enzyme has other functions inside the
Reaction rate 2 reaction rate 2 cell.
Km 21.18
Km 24.25
1 Vmax 3.585 1 Vmax 3.423 • MDH is present in two forms, homo-dimeric and tetrameric. In Gram negative
bacteria, it present as dimer while tetramer in Gram positive bacteria and
0 0 archaea.
0 50 100 150 0 50 100 150
OAA conc. (uM) NADH conc. (uM)
• The alarmone ppGpp is involved in growth regulation and various stress
• MDH purity analyzed by SDS PAGE and visualize by Coomassie Brilli responses in bacteria. It induces profound transcriptional alterations, including
ant Bluestaining (Figure A). the repression of stable RNA synthesis and induction of stress response factors
• MDH activity assay designed and analyze with and without ppGpp to p and genes required for amino acid biosynthesis and transport.
robe and found no significant effect of ppGpp on MDH activity (Figure
B, C). • In this study, we report MDH as a novel ppGpp-binding protein with a high
• Km and Vmax determined for oxaloacetate and NADH, values calculat affinity. No activity change associated with the ppGpp-binding suggests that
ed using GraphPad Prism 8 software (Figure D, E) ppGpp does not bind at active site of MDH but MDH act as a reservoir for
ppGpp with no effect to its function, for which crystal structure of MDH bound
to ppGpp will bey a key to open a new dimension.
