A thesis for the Degree of Master

Biochemical characterization of glyceraldehyde-3-

phosphate dehydrogenase from Thermococcus

kodakaraensis KOD1

By

Le Thuy Linh

GYEONGSANG NATIONAL UNIVERSITY

DIVISION OF APPLIED LIFE SCIENCE

2009

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Biochemical characterization of glyceraldehyde-3-

phosphate dehydrogenase from Thermococcus

kodakaraensis KOD1

A Dissertation submitted to Faculty of the Graduate School

of the Gyeongsang National University

By Le Thuy Linh

In partial fulfillment of the requirements

for the degree
of Master of Science

May, 2009

Dr. Gang-Won Cheong, Dissertation Supervisor

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 CONTENTS

Contents ...................................................................................................................i

List of figures ...........................................................................................................iii

List of table ..............................................................................................................iv

Abbreviations ...........................................................................................................v

..........................................................................................................................vi

Abstract ....................................................................................................................vii

I. Introduction...........................................................................................................1

II. Materials and methods.........................................................................................4

1. Organism ........................................................................................................4

2. Chemical and reagents....................................................................................4

3. Thermococcus kodakaraensis KOD1 culture procedure ................................4

4. Polymerase chain reaction (PCR)...................................................................5

5. Cloning and expression of GAPDH-tk ...........................................................5

6. Purification of GAPDH-tk..............................................................................6

7. Site-directed mutagenesis of GAPDH-tk........................................................7

8. Enzyme assay .................................................................................................7

9. Kinetic study...................................................................................................8

10. Heat stability tests.........................................................................................8

11. Treatment of GAPDH-tk with oxidative stress .............................................9

12. Electron microscopy and image processing..................................................9

III. Results and discussion .......................................................................................11

1. Alignment of GAPDH-tk sequence with homologs .......................................11

2. Cloning and expression of GAPDH-tk ...........................................................15

 3. GAPDH-tk purification ..................................................................................17

4. GAPDH-tk activity determination ..................................................................20

5. Kinetic properties ...........................................................................................26

6. Structure of GAPDH-tk ..................................................................................28

7. Response of GAPDH-tk under oxidative stress.............................................. 31

IV. Conclusion..........................................................................................................39

V. References ...........................................................................................................40

Acknowledgement ...................................................................................................45

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 List of figures

Figure 1: Multiple amino acid alignment of GAPDH homologues ......................................... 13

Figure 2: Recombinant pET28a-GAPDH-tk confirmation ...................................................... 15

Figure 3: GAPDH-tk expression in E. coli BL21 (DE3) ......................................................... 15

Figure 4: GAPDH-tk purification by Ni-NTA column ............................................................ 17

Figure 5: GAPDH-tk analysis by gel filtration chromatography ............................................. 18

Figure 6: Effects of pH and temperature on purified GAPDH-tk activity ............................... 20

Figure 7: Thermostability of GAPDH-tk at 80 0C and 90 0C................................................... 21

Figure 8: Glycolytic activity of GAPDH-tk wide type (GAPDH-tk-WT) and GAPDH

mutant (GAPDH-C141S) ......................................................................................................... 23

Figure 9: Expression and purification of GAPDH-C141S....................................................... 23

Figure 10: GAPDH-C141S analysis by gel filtration chromatography ................................... 24

Figure 11: Electron micrograph and image processing of the negatively

stained GAPDH-tk ................................................................................................................... 29

Figure 12: Oxidized GAPDH-tk forms were visualized on 7.5 % native gel .......................... 33

Figure 13: GAPDH-tk proteins treating without or with H2O2, NO were separated by gel
filtration chromatography ........................................................................................................ 34

Figure 14: GAPDH-tk proteins treating without or with H2O2, NO were observed by
transmission electron microscopy ............................................................................................ 36

Figure 15: Glycolytic activity test for oxidized GAPDH-tk forms. ......................................... 37

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 List of table

Table 1: Kinetic parameters for oxidation reaction of GAPDH-tk .......................................... 26

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 Abbreviations

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

D-G3P D-glyceraldehyde-3-phosphate

NAD+ Nicotinamide adenine dinucleotide

SNP Sodium nitroprusside

SE Standard error

Ni-NTA Nickel nitrilotriacetic acid

FPLC Fast protein liquid chromatography

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IPTG Isopropyl - D - thiogalactopyranoside

NCBI National center for biotechnology information

PCR Polymerase chain reaction

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TEM Transmission electron microsopy

NO Nitric oxide

TEMED N,N,N',N'-tetramethylethylenediamine

EM Electron microscopy

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) NAD+ cofactor

D-glyceraldehyde 3-phosphate (D-G3P) 1,3-diphosphoglycerate

glycolysis . ,

GAPDH oxidative stress glycolytic function ,

endocytosis, apoptosis, phosphotransferase, nuclear RNA export, DNA repair

GAPDH

. GAPDH homotetramer .

Thermococcus kodakaraensis KOD1 GAPDH

GAPDH gene clone , Ni-NTA column

. GAPDH-tk subunit ~37 kDa, Gel filtration

chromatography native polyacrylamide gel electrophoresis native

GAPDH-tk 145 kDa . transmission electron

microscopy (TEM) image processing program GAPDH-tk tetramer

. GAPDH-tk oxidative stress (~232 kDa)

form gel-filtration TEM .

GAPDH-tk 80 0C , half-life 5

. Mutant (GAPDH-C141S) Cysteine 141 glycolytic

active site , Cysteine

141 GAPDH oligomer .

GAPDH-tk NAD+ D-G3P Km

77.8 7.5 M 49.33.0 M , Vmax 45.1 0.8 U/mg

59.6 1.3 U/mg. GAPDH-tk GAPDH

, activity .

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 ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in

glycolysis by catalyzing the reversible reaction of D-glyceraldehyde-3-phosphate (D-

G3P) into 1,3-diphosphoglycerate using NAD+ as a cofactor. However, a variety of recent

studies are now suggesting that GAPDH is a multifunctional protein. GAPDH has non-

glycolytic function under a lot of stressors, most of which are associated with oxidative

stress. All GAPDH homologs thus far have a tetramer. In our report, we cloned and

overexpressed GAPDH from hyperthermophilic archaeon Thermococcus kodakaraensis

KOD1 (GAPDH-tk). GAPDH-tk was purified by using Ni-NTA column. Molecular mass

of one subunit of GAPDH-tk was approximately 37 kDa. Gel filtration chromatography

and native polyacrylamide gel electrophoresis determined that the native molecular

weight of GAPDH-tk was 145 kDa, approximately. In addition, transmission electron

microscopy (TEM) and image processing showed that GAPDH-tk had tetrameric

structure. Interestingly, GAPDH-tk appeared a new form with high molecular weight

(~232 kDa) under oxidative stress. Oxidized GAPDH-tk forms were separated by gel

filtration chromatography and observed by TEM. Furthermore, glycolytic activity of

wild-type GAPDH and mutant GAPDH-C141S was measured. The results indicated that

cysteine 141 was as active site of GAPDH-tk. Under the assay conditions, the optimal

value of pH and temperature for GAPDH-tk activity were 7.0 and 80 0C, respectively.

GAPDH-tk exposed high activity at 80 0C. GAPDH-tk was thermo-stable protein with a

half-life of 5 hours at 80 0C and 90 0C. The kinetic parameters of GAPDH-tk were

calculated by using Lineweaver-Burk plots. The apparent Km values for NAD+ and D-

G3P were 77.8 7.5 M and 49.3 3.0 M with Vmax values of 45.1 0.8 U/mg and

59.6 1.3 U/mg, respectively.

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 I. INTRODUCTION

Since hyperthermophilic archaea have survived in extreme environments and

have uniquely metabolic and physiological characteristics, they are used widely in the

biotechnological industry for diverse applications [1, 2, 3]. T. kodakaraensis KOD1 was

isolated from a solfatara on the shore of Kodakara Island, Kagoshima, Japan [4]. They

live at high temperature from 60 0C to 100 0C (optimum 85 0C) with pH range 5.0-9.0. T.

kodakaraensis KOD1 can be regarded as one of the most useful model organisms in the

research on hyperthermophiles.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme of the

glycolytic pathway that catalyzes the phosphorylation of glyceraldehyde-3-phosphate to

1,3-bisphosphoglycerate. The reaction catalyzed by GAPDH is really the sum of two

processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of

the carboxylic acid and orthophosphate to form the acyl-phosphate product. This enzyme

occurs universally throughout the bacterial, eucaryal, and archaeal kingdoms.

GAPDH has been studied in many organisms including archaea. The archaeal

GAPDHs share less identity with bacterial and eukaryotic enzymes (16%-20%), and

display dual cofactor specificity for NAD+ and NADP+ [5], in contrast to the bacterial and

eukaryal enzymes, which are usually specific for NAD+ [6, 7]. The D-glyceraldehyde-3-

phosphate dehydrogenase from Methanothermus fervidus reacted with both NAD+ and

NADP+ and was not inhibited by pentalenolactone [8]. Sulfolobus solfataricus GAPDH

was able to use both NAD+ and NADP+, but exhibited a marked preference for NADP+

[9].

However, recent evidences demonstrate that GAPDH is a multifunctional protein

[10]. These included roles for GAPDH in membrane transport and membrane fusion,

microtubule assembly, phosphotransferase activities, nuclear RNA export, DNA

replication and DNA repair. Each activity appears to be distinct from its glycolytic

function. Other independence studies implicated GAPDH as an essential part of the

program of gene expression observed in apoptosis and as part of the cellular phenotype of

age-related neuronal disorders and other properties [10].

In addition to the various functions of GAPDH described above, GAPDH

frequently associated with oxidative stress. Nitric oxide (NO) is one of the major cellular

signaling molecules and is known to mediate cell death. GAPDH had received particular

attention as one of the major targets of NO in cells after the discovery of NO-induced

ADP ribosylation of GAPDH, inhibiting its glycolytic activity [11, 12]. The glycolytic

enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-

sensitive cysteine residue may provide additional input signals. In response to H2O2 stress,

GAPDH from Schizosaccharomyces pombe is transiently oxidized at Cys-152, which

enhances the association of GAPDH with Mcs4 response regulator [13]. The nuclear

translocation of human GAPDH during oxidative stress constitutes a protective

mechanism to safeguard the genome by preventing structural inactivation of APE1, an

essential enzyme involved in the repair of abasic sites in damaged DNA [14].

Although GAPDH characterizations have previously been examined in

thermophilic archaeon such as S. solfataricus, M. fervidus and mesophilic methanogenic

archaebacteria Methanobacterium bryantii, Methanobacterium formicicum, the non-

glycolytic properties from archaeal GAPDH has not found. Thus, we cloned and

overexpressed GAPDH from T. kodakaraensis KOD1 (GAPDH-tk). To determine

function of GAPDH-tk, GAPDH-tk was purified and then measured glycolytic activity.

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Kinetic parameters of GAPDH-tk were calculated to compare activity of GAPDH-tk with

GAPDHs from other organisms. Moreover, mutant GAPDH-C141S was also purified and

tested activity to indicate the active site of GAPDH-tk. Then, quaternary structure of

GAPDH-tk was observed by transmission electron microscopy and image processing.

Lastly, we tested association of GAPDH-tk with oxidative stress. We found that GAPDH

underlies oxidative stress-induced aggregation. Furthermore, oxidized GAPDH has lost

glycolytic activity. This is the first description of oxidized-GAPDH form from archaea.

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 II. MATERIALS AND METHODS

1. Organism

T. kodakaraensis KOD1 which is kindly donated by Japan Collection of

Microorganisms, RIKEN BioResource Center, Japan, was used to prepare crude cell

extracts and isolate genomic DNA. It was cultured in the 280 Thermococcus medium [15].

2. Chemical and reagents

Most chemicals were purchased from Sigma, Amresco and domestic companies.

Electrophoresis agents such as acrylamide, Sodium lauryl sulfate (SDS), tris-base,

glycine and N,N-methylene-bis-acrylamide sodium thiosulfate, silver nitrate and sodium

carbonate for silver staining were purchased from Sigma.

For the cloning and expression, Taq-polymerase, dNTP in polymerase chain

reaction process were purchased from Takara. And pET28a(+) expression vector was

from Invitrogen. Chromatographic support Ni-NTA His-bind resins were from QIAGEN

and the superdex 200 column for gel filtration was from GE Healthcare Life Sciences.

3. T. kodakaraensis KOD1 culture procedure.

Media were prepared from the medium 280 including KCl 0.33 g, MgCl26H2O
2.8 g, MgSO47H2O 3.4 g, NH4Cl 0.25 g, NaCl 25.0 g, K2HPO4 0.3 g, Yeast extract (Difco)

3.0 g, Tryptone (Difco) 3.0 g, FeSO47H2O 0.025 g, NaBr 10.0 mg, Trace minerals 10.0

ml, Vitamin solution 10.0 ml, Sulfur (powder) 10.0 g, Na2S9H2O 0.5 g, Resazurin 1 ml,

distilled water 1 L. Ingredients were mixed, except sulfur and Na2S9H2O, and adjusted to

pH 7.0-7.2. Then, media were autoclaved. Separately, sulfur was steamed at 90 0C for 3
hours in shaking water bath. Na2S9H2O as a 5 % solution was neutralized and autoclaved.

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After that, sulfur and Na2S9H2O 5 % solution were added into the media and then

distributed to culture vessels under a N2 stream seal with butyl rubber stoppers.

T. kodakaraensis KOD1 cells was cultured under anaerobic condition at 80 0C

for 6 hours in sharking water bath.

4. Polymerase chain reaction (PCR)

Primer sequences:

Forward: 5-GGA ATT CCA TAT GAA GGT GAA AGT C-3

Reverse: 5-CCG CTC GAG TCA CTT CAG AAT TCC AA -3

Genomic DNA isolated from T. kodakaraensis KOD1 was used as template, the

mixture reaction including 1 l DNA template, 1 l forward primer, 1 l reverse primer,

0.5 l pfu DNA polymerase, 4 l dNTPs, 10 l Taq buffer and 32.5 l distiller water. The

PCR reaction was performed for 33 cycles with denaturation at 94 oC for 20 seconds,

annealing at 50 0C for 10 seconds and extension at 72 0C for 2 minutes 15 seconds. For

the analysis of PCR products, 2 l of PCR product was electrophoresed on 0.8 % agarose

gel and visualized in ultraviolet radiation by staining with ethidium bromide.

5. Cloning and expression of GAPDH-tk

PCR product and pET28a(+) expression vector were digested by restriction

endonucleases Xho I and Nde I. Then, digested PCR product and pET28a(+) expression

vector were ligated and cloned to E. coli XL1 Blue. The cells were cultured at 37 0C for 6

hours. The recombinant plasmids were purified and then transformed to E. coli BL21

(DE3) competent cell for protein expression.

E. coli BL21(DE3) cells containing recombinant pET28a-GAPDH-tk plasmid

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were cultured in 2 liters of LB broth media with 30 g/ml Kanamycine at 37 0C for 3

hours. When OD600 reached at 0.7, isopropyl D thiogalactopyranoside (IPTG) was

added to induce the protein expression. The culture was shaken over an induction period

of 4 hours. The cells were harvested by centrifugation at 6000 rpm for 10 min. The cells

were resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM

2-mercaptoethanol, 20 mM imidazole. The suspensions were sonicated and heated at 65

0
C for 1 hour. Then, the thermo-stable supernatants were collected by centrifugation. The

crude proteins were subsequently applied to protein purification system.

6. Purification of GAPDH-tk

Crude proteins were loaded on Ni-NTA column. Recombinant GAPDH-tk

proteins have 6xHig-tagged that binds to column. The unbound proteins flowed through

column. The column was washed by lysis buffer. The GAPDH-tk proteins were eluted by

different concentrations of imidazole in a range of 50 mM-250 mM. The purified

GAPDH-tk proteins were visualized on 12.5% SDS-PAGE. Then, the eluted proteins

were dialysed against 100 mM imidazole buffer containing 50 mM Tris, 300 mM NaCl,

20 mM 2-mercaptoethanol.

Continuously, the purified GAPDH-tk proteins were analysed by gel filtration

chromatography. Gel filtration chromatography was performed using Superdex 200

10/30 column (GE Healthcare) on FPLC system (BIO-RAD) equilibrated by 0.2 m

filtered Tris buffer (50 mM Tris, 300 mM NaCl, 20 mM 2-mercaptoethanol; pH 8.0) at a

flow rate of 0.4 ml/min at 25 C. Protein peaks were detected at 280nm wavelength.

Standard proteins included thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232

kDa), aldolase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa). Collected protein

samples were checked on SDS-PAGE and Native-PAGE.

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 7. Site-directed Mutagenesis of GAPDH-tk

GAPDH-tk has one cysteine at amino acid position 141. The complementary

primer pairs were designed as follows:

Forward: 5-GTC GTC TCC AGT AAC ACG ACT -3

Reverse: 5-AGT CGT GTT ACT GGA GAC GAC -3

Recombinant pET28a-GAPDH-tk plasmid was used as DNA template, the

mixture reaction including 1 l DNA template, 0.5 l forward primer, 0.5 l reverse

primer, 1 l pfu DNA polymerase, 4 l dNTPs, 10 l 5x pfu DNA polymerase buffer and

33 l distill water. The PCR reaction was performed for 18 cycles with denaturation at 94
o
C for 30 seconds, extension at 95 0C for 30 seconds, 55 0C for 1 minute and 68 oC for 12

minutes. The PCR reaction was cooled to room temperature and then 1 l of restriction
o
enzyme DpnI was added. The mixture was incubated at 37 C for 15 hours.

Transformation of 5 l of reaction into competent E. coli BL21 (DE3) was carried out at

42 0C for 1 min. The mutant was confirmed by DNA sequencing. The GAPDH-C141S

proteins were purified by the same methods with wild type purification.

8. Enzyme assay

The enzyme activity was measured by following the increase in absorbance at

340 nm at 80 0C using a Libra S12 (biochrom) spectrophotometer. The standard assay

mixture (total volume 1.0 ml) contained 50 mM Tris/HCl, 20 mM 2-mercaptoethanol, 10

mM potassium arsenate, 10 mM D-glyceraldehyde-3-phosphate (D-G3P) and 1mM

NAD+ (pH 8.0 adjusted at 20 0C; resulting pH 7.0 at 80 0C). Since glyceraldehyde-3-

phosphate is a thermolabile substrate, it was added just before the reaction was started by

the enzyme. Unlike D-G3P, NAD+ was stable up to 70 0C [8].

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 The enzyme activity was determined from the initial velocity of the reaction.

One unit GAPDH-tk activity is defined as the amount of enzyme which catalyzes the

formation of 1 mol NADH/min under the condition used. Specific activity of the

enzyme is the ratio of enzyme activity to amount of protein in enzyme assay.

Although NADH is not stable at higher temperatures and therefore the

measurement of NADH-producing enzymatic reactions will tend to underestimate the

enzyme activities, there is only a 0.4 % difference between the directly measured

absorption increase and the absorption increase corrected for NADH decay up to 70 0C

[8].

9. Kinetic study

For kinetic studies, initial velocities of the enzymatic reaction were carried out

by varying the concentration of the substrates, D-G3P (from 0.04 mM to 10 mM) or

NAD+ (from 0.02 mM to 2 mM). Values of the Michaelis constants (Km), dissociation

constants (Kcat), and maximal velocity (Vmax) were obtained by mathematical calculation

according to the method of Cleland [16]. Protein concentrations were estimated by the

method of Bradford [17] using bovine serum albumin (BSA) as a standard.

10. Heat stability tests

The temperature stability of GAPDH was investigated by incubating GAPDH

solution (0.11 mg/ml; basic incubation buffer: 50 mM Tris/HCl, 20 mM 2-

mercaptoethanol, 10 mM potassium arsenate; total volume 80 l) at two temperatures (80

0
C and 90 0C) with time range from 1 hour to 8 hours. Then, the samples were centrifuged

briefly and tested for enzyme activity at 80 0C directly.

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 11. Treatment of GAPDH-tk with oxidative stress

GAPDH-tk proteins were treated with 1 mM H2O2 and 1 mM sodium

nitroprusside (SNP), a NO donor. The mixtures were incubated at 37 0C and 80 0C for 1

hour, 5 hours, 10 hours. After treatment, the proteins were analyzed by native PAGE. The

native gel was stained by Coomassie Blue.

The oxidized GAPDH-tk forms were separated by using gel filtration

chromatography. The molecular weight of oxidized GAPDH-tk was determined by SDS

gel and native gel. Finally, the oxidized GAPDH-tk forms were observed by TEM.

12. Electron microscopy and image processing

The purified GAPDH-tk proteins in 50 mM TrisHCl (pH 8.0) containing 300

mM NaCl, 20 mM 2-mercaptoethanol were applied to glow-discharged carbon coated

copper grids. Allowing the protein to adsorb for 12 min, the grids were rinsed with

deionized water and stained with 2% (w/v) uranyl acetate. Specimens were examined in

the FEI Technai-12 at an acceleration voltage of 120 kV using a low-dose unit [18].

Electron micrographs (EM) were recorded on Kodak film (SO163) at a nominal

magnification of 67,000. Light-optical diffractograms were used to select the

micrographs, to examine the defocus, and to verify that no drift or astigmastism was

present. Suitable areas were digitized as arrays of 1024 1024 pixels with Leaf Scan 45

at a pixel size of 20 m corresponding to 0.30 nm at specimen level. For image

processing, the SEMPER [19] and EM [20] software packages were used. From digitized

micrographs, smaller frames of 32 32 pixels containing individual particles were

extracted interactively. These images were aligned translationally and rotationally using

standard correlation methods [21] and [22]. For analyzing the rotational symmetry of top-

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on view images, the aligned images were subjected to multivariate statistical analysis [23].

The individual images were aligned translationally but not rotationally as described by

Marco et al. [24]. The resulting eigenimages represent all important structural features of

the original data set. If the images have different rotational symmetries in the original

data set, the eigenimages reveal the different symmetry axes. Moreover, these images can

be distinguished and subsequently separated based on eigenimages. The rotationally

aligned images were classified based on eigenvectoreigenvalue data analysis,

subsequent averaging was performed for each class separately.

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 III. RESULTS AND DISCUSSION

1. Alignment of GAPDH-tk sequence with other homologs

DNA sequence coding GAPDH-tk comprised an open reading frame of 1005

base pairs; predicting GAPDH-tk composed 334 amino acids with a molecular mass ~ 37

kDa. GAPDH shares two conserved domains: a Rossmann-fold NAD(P)H/NAD(P)(+)

binding (NADB) domain and C-terminal catalytic domain. The NADB domain is found

in numerous dehydrogenases of metabolic pathways such as glycolysis, and many other

redox enzymes. NAD binding involves numerous hydrogen-bonds and van der Waals

contacts, in particular H-bonding of residues in a turn between the first strand and the

subsequent helix of the Rossmann-fold topology. Characteristically, this turn of GAPDH-

tk exhibits GYGTIG, a consensus binding pattern, in which the first 2 glycines participate

in NAD(P)-binding, and the third facilitates close packing of the helix to the beta-strand.

The C-terminal catalytic domain is a mixed /antiparallel fold. GAPDH-tk conserved

only one cysteine 141 like active site at C-terminal catalytic domain.

GAPDH homologs have been searched by BLAST in NCBI database. The

alignment was generated using Clustal W program. Resulting that identical residues in

black are reflected at least 60% identity of archaeal GAPDHs. Eukaryotic GAPDHs share

less identity (20%) (fig. 1).

Due to the high sequence similarity between GAPDH-tk and GAPDH from

hyperthermophilic archaeon S. solfataricus (GAPDH-ss), the structure of GAPDH-tk will

be compared to that of GAPDH-ss. GAPDH-ss subunit has two folding domains, the

nucleotide-binding domain (residues 1-138 and 301-333) and the / fold catalytic

domain (residues 139-300 and 334-340) build around a mainly antiparallel -sheet [25].

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The nucleotide-binding domain of GAPDH-ss consists of seven -sheets and eight -

helixes with a conserved double ---- motif fold. GAPDH-ss has three cysteine

residues, one cysteine at nucleotide-binding domain and two cysteines at catalytic domain.

The active site cysteine 139 of GAPDH-ss at catalytic domain is located at the C-

terminus of -helix 138-153. Cysteine 141 of GAPDH-tk conserved in the same

topological position as active site cysteine 139 of GAPDH-ss. The substrate phosphate

and inorganic phosphate binding of GAPDH-ss are serine 138, asparagines 140, arginine

166 and arginine 167. These residues also conserved in GAPDH-tk sequence.

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 Figure 1: Multiple amino acid alignment of GAPDH homologues.

The original sequences of GAPDH were aligned from T. kodakaraensis KOD1

(YP 183178), S. solfataricus (CAA 47040), Pyrococcus woesei (P 61880), M. formicium

(CAB 37402), M. fervidus (1CF2Q), Homo sapiens (human) (P04406), Rattus norvegicus

(norway rat) (P04797) and S. pombe (P78958). Archaeal GAPDH sequences were shown

in the first five organisms. Eukaryotic GAPDH sequences were shown in the last three

organisms. The black line upper sequences indicated the Rossmann-fold

NAD(P)H/NAD(P)(+) binding (NADB) domain. The broken-black line upper sequences

indicated C-terminal catalytic domain. A consensus binding pattern, GYGTIG, conserved

in NADB domain was underlined. The cycle indicated residues of GAPDH-tk enzyme in

the substrate phosphate and inorganic phosphate binding. The star implicated residue in

the catalytic mechanism.

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2. Cloning and expression of GAPDH-tk

TK0765 gene coding GAPDH was isolated from T. kodakaraensis KOD1

genome. This gene was amplified by PCR. The PCR product strains have nearly 1000

base pairs visualized in 0.8 % agarose gel (fig. 2, lane 3). Cloning method was described

in Materials and methods. The ligation and cloning were confirmed (fig. 2). pET28a-

GAPDH-tk plasmids (fig. 2, lane 1) were selected and transformed to E. coli BL21 (DE3)

for protein expression. The E. coli BL21 (DE3) cells containing recombinant pET28a-

GAPDH-tk plasmid were firstly cultured with small amount. When concentration of cells

reached OD600 = 0.7, IPTG was added and continuously cultured for more 4 hours.

GAPDH-tk expression was checked on SDS-PAGE (fig. 3). The gel was stained by

Coomassie Blue. The increased bands indicated high level expression of GAPDH protein

corresponding with molecular weight of subunits around 37 kDa.

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 M 1 2 3 4

bp

23130
9416
6557
4361

2322
2027

564

Figure 2: Recombinant pET28a-GAPDH-tk confirmation.

Lane M: DNA marker; lane 1, 2: digested pET28a-GAPDH-tk vector; lane 3: PCR

product; lane 4: digested pET28a.

kDa 1 2 3 4 5 6

66

45

36

29

24

Figure 3: GAPDH-tk expression in E. coli BL21 (DE3).

Lane 1: total proteins from non-induced cells; lane 2-6: total proteins from IPTG-
induced cells.

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3. GAPDH-tk purification.

The crude proteins from IPTG-induced cells were firstly heated at 65 0C for 1

hour. The other proteins except GAPDH-tk were denatured and aggregated at high

temperature. After heating, the mixture was centrifuged at 10.000 rpm for 10 min. The

thermo-stable supernatant was collected to prepare protein purification.

Ni-NTA affinity chromatography was used to purify 6xHis-tagged recombinant

proteins. The thermo-stable supernatant was loaded into Ni-NTA column. The unbound

proteins flowed out and the column was washed by lysate buffer. GAPDH-tk proteins

were eluted by concentrations of imidazole in range 50 mM - 250 mM. Purity of

GAPDH-tk was visualized on 12.5 % SDS-PAGE (fig. 4). The molecular mass of

subunits of GAPDH-tk is approximately 37 kDa. Unlike GAPDHs from M. fervidus (~ 45

kDa) [8], molecular mass of subunits of GAPDH-tk is similar with hyperthermophilic

archaeon S. solfataricus (~ 39 kDa) [9] and eucaryal GAPDH such as rabbit GAPDH (~

36 kDa) [27], D-GAPDH from European pilchard Sardina pilchardus (~ 37 kDa) [28].

The native molecular weight of GAPDH-tk proteins were determined by gel

filtration chromatography and native PAGE (fig. 5). There is one peak absorbance at 280

nm (fig. 5A). Purified GAPDH-tk fractions from this peak were analyzed by native PAGE

and SDS-PAGE (fig. 5B). GAPDH-tk has native molecular weight ~ 145 kDa. These

results indicated that GAPDH-tk is a tetramer of 37 kDa subunits, which is the same with

other GAPDHs in both achaea and eucarya [8, 9, 27, 28].

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 kDa
1 2 3 4 5 6 7 8 9 10

100
70

50
40

30

20

Figure 4: GAPDH-tk purification by Ni-NTA column.

Lane 1, protein marker. Lane 2, crude proteins from non-induced cells; lane 3,

crude proteins from IPTG-induced cells; lane 4, soluble extract after heating at 65 0C for

1 hour; lane 5, unbound proteins after loading Ni-NTA column; lane 6, washed proteins

by lysate buffer (50 mM Tris, 300 mM NaCl, 20 mM 2-mercaptoethanol, 20 mM

imidazole); lane 7-10: eluted proteins by 50 mM imidazole, 100 mM imidazole, 250 mM

imidazole in lysate buffer.

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 A
0.45 440
660 232 140 43
0.4
a bsor ban c e at 2 80n m 0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 10 20 30 40
Time (min)

kDa M 30 31 32 33 34 35
B 669

440

232

140

67

Figure 5: GAPDH-tk analysis by gel filtration chromatography.

(A) Purified GAPDH-tk was loaded on superdex 200 10/300 GL column. (B)

Different fractions from gel filtration chromatography were loaded on 7.5% native gel.

SDS-PAGE was shown in below panel. Lane M shows the protein marker. The protein

fractions were indicated by the number from 30 to 35.

- -
19
4. GAPDH-tk activity determination

GAPDH catalyzed the reversible reaction glyceraldehyde-3-phosphate to 1,3-

bisphosphoglycerate in the present of NAD+. This reaction generated NADH. Thus, the

enzyme activity was determined spectrophotometrically at 80 0C by monitoring NADH

generation at 340nm.

Since glyceraldehyde-3-phosphate is a high instability of substrate with a half-

life of 6 min at 70 0C [6], the enzyme investigations are limited. Therefore, the oxidative

reaction was performed by applying high glyceraldehydes-3-phosphate concentration (10

mM), which guaranteed substrate saturation of the enzyme during measurement.

To determine assay condition, the influence of pH and temperature on purified

GAPDH-tk activity was tested. Specific activity of purified GAPDH-tk was examined in

the pH range 6.2 - 9.0 using a mixture of different buffers such as sodium phosphate,

HEPES, Tris (fig. 6A). The optimal value of pH was approximately 7.0 which consisted

with pH range 5.0-9.0 for growth of T. kodakaraensis KOD1. The optimal value of pH of

GAPDH-tk is different with hyperthermophilic GAPDH from M. fervidus (optimal pH ~

8.5) [8] due to difference of environmental condition of living organisms. GAPDH-tk

activity was measured at several temperatures from 30 0C to 90 0C (fig. 6B). GAPDH-tk

exposed high activity in temperature range 70 0C 90 0C while GAPDH from S.

pilchardus (GAPDH-sp) exposed high activity in low temperature range 28 0C 32 0C

[28]. Because S. pilchardus is pilchard which is small pelagic fishes of the north-west

coast of Africa, GAPDH-sp act at lower temperature than GAPDH-tk.

- -
20
 A 7

5
Specific activity (U/mg)

0
6 6.5 7 7.5 8 8.5 9 9. 5
pH

B
7

5
Specific activity (U/mg)

0
0 10 20 30 40 50 60 70 80 90 100
Temperature ('C)

Figure 6: Effects of pH and temperature on purified GAPDH-tk activity.

(A) Specific activity of purified GAPDH-tk in the pH range 6.2 9.0 using a

mixture of different buffers. (B) GAPDH-tk activity was measured at several

temperatures from 30 0C to 90 0C. Values are given as means of three separate

experiments.

- -
21
 Thermostability of GAPDH-tk was checked at 80 0C and 90 0C (fig. 7). The

proteins were incubated from 1 hour to 8 hours before testing activity. The result shows

that GAPDH-tk is thermo-stable protein with a half-time of 5 hours at 80 0C and 90 0C.

But GAPDH-tk has shorter activity half-time than hyperthermophilic GAPDH from S.

solfataricus at 80 0C for 17h [9]. The half-time of GAPDH from P. woesei also reported

to be 65 min at 97 0C [29]. Moreover, several salts were able to stabilize GAPDH enzyme,

especially at high ionic strength, at 90 0C for 30 min [8].

10

7
specific activity (U/mg)

6
80 'C
5
90 'C
4

0
0 1 2 3 4 5 6 7 8 9
Time (h)

Figure 7: Thermostability of GAPDH-tk at 80 0C and 90 0C.

- -
22
 The active site of GAPDH has been reported as cysteine 139 from S. solfataricus

and cysteine 151 from B. stearothermophilus [25], cysteine 150 from rat and cysteine 152

from human [26]. GAPDH-tk protein has only one cysteine at amino acid position 141.

To determine catalytic site of GAPDH-tk, the mutant GAPDH-tk (GAPDH-C141S) was

also purified by the same methods with wild type GAPDH-tk (fig. 9). Glycolytic activity

was measured at 80 0C, pH 7.0 for GAPDH-tk and GAPDH-C141S (fig. 8). GAPDH-

C141S has much lower activity than GAPDH-tk. The result indicates that cysteine 141 is

as active site of GAPDH-tk.

On the other hand, purified GAPDH-C141S proteins were analyzed by gel

filtration chromatography (fig. 10). One peak was detected at 280 nm (fig. 10A). The

native molecular of GAPDH-C141S proteins was visualized on native PAGE and SDS-

PAGE (fig. 10B). GAPDH-C141S formed tetramer (~ 145 kDa) with molecular mass of

subunit ~ 37 kDa. The results demonstrate that tetrameric structure of GAPDH-tk is not

formed by disulfide bond. In contract to GAPDH from S. solfataricus (GAPDH-ss), a

disulphide bridge holds together an -helix 138 - 153 from the catalytic domain and a

short -helix 120 - 125 from the nucleotide-binding domain on the surface of the tetramer

[25].

- -
23
 110

100

90

80
relative activity (%)
70

60 GAPDH-tk-W T
50 GAPDH-C141S

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9
Time (min)

Figure 8: Glycolytic activity of GAPDH-tk wide type (GAPDH-tk-WT) and GAPDH

mutant (GAPDH-C141S).

A B

kDa 1 2 3 4 5
kDa 1 2 3 4 5 6 7

66
66
45
45
36
36

29
29 24
24
20 20

Figure 9: Expression and purification of GAPDH-C141S.

(A) GAPDH-C141S expressed in E. coli BL21 (DE3). Lane 1, protein marker;

lane 2: crude proteins from non-induced cells; lane 3-5: crude proteins from IPTG-

induced cells. (B) GAPDH-C141S was purified by Ni-NTA column. Lane 1, protein

marker; lane 2, soluble extract after heating at 65 0C for 1 hour; lane 3, unbound proteins

after loading Ni-NTA column; lane 4, washed protein by lysate buffer; lane 5-7: eluted

protein by 50 mM imidazole, 100 mM imidazole, 250 mM imidazole in lysate buffer.

- -
24
 A
0.09
660 440 232 140 43
0.08

Absorbance at 280nm 0.07

0.06

0.05

0.04

0.03

0.02

0.01

0
0 5 10 15 20 25 30 35 40
Time (min)

B kDa M 26 27 28 29 30 31 32 33 34 35
669
440

232

140

67

Figure 10: GAPDH-C141S analysis by gel filtration chromatography.

(A) Purified GAPDH-C141S was loaded on superdex 200 10/300 GL column.

(B) Different fractions from gel filtration chromatography were loaded on 7.5% native gel.

SDS-PAGE was shown in below panel. Lane M shows the protein marker. The protein

fractions were indicated by the number from 26 to 35.

- -
25
 5. Kinetic properties

GAPDH-tk catalyses a two-substrate reaction, the Km values for NAD+ and D-

glyceraldehyde-3-phosphate (D-G3P) were determined by varying the concentration of

one of the substrate (D-G3P or NAD+) and keeping a constant concentration of the other.

The final value of kinetic parameters was the mean of three determinations S.E. The Km

values for NAD+ and D-G3P were 77.8 M and 49.3 M, respectively (table 1). The Vmax

NAD+ and D-G3P were calculated to be 45.1 U/mg and 59.6 U/mg, respectively.

We compare kinetic parameters of T. kodakaraensis KOD1 with other organisms,

which published in previous papers [8, 9, 28]. The Michaelis constant, Km, has two

significant meaning for kinetic property. Firstly, Km provides a measure of the substrate

concentration required for significant catalysis to occur. Secondly, Km is a measure of the

strength of the enzyme-substrate complex: a high Km indicates weak binding; a low Km

indicates strong binding. It must be stressed that Km indicates the affinity of the enzyme-

substrate complex when rate constant k-1 (dissociation of enzyme and substrate) is much

greater than k2 (proceed to form product). In comparison with GAPDH from M. fervidus,

S. solfataricus and S. pilchardus, the Km of NAD+ and D-G3P of T. kodakaraensis KOD1

is lower value than the others. Thus, the binding of NAD+ and D-G3P to GAPDH-tk is

stronger than the others. The maximal rate, Vmax, is attained when the catalytic sites on

the enzyme are saturated with substrate. The Vmax of NAD+ of T. kodakaraensis KOD1 is

highest that leads to exhibit high activity of GAPDH-tk.

On the other hand, Vmax reveals the turnover number (Kcat) of an enzyme, which

is the number of substrate molecules converted into product by an enzyme molecule in a

unit time when the enzyme is fully saturated with substrate. Kcat of T. kodakaraensis

KOD1 is 9.8 s-1.

- -
26
GAPDH Kinetic parameters Km NAD+ Km D-G3P Vmax NAD+ Vmax D-G3P Kcat
from other organisms (M) (M) (U/mg) (U/mg) (s-1)
GAPDH-Thermococcus kodakaraensis 77.8 7.5 49.3 3.0 45.1 0.8 59.6 1.3 9.8 1.0
KOD1
GAPDH-Methanothermus fervidus a 650 a - 12 a - -

GAPDH-Sulfolobus solfataricus a 2200 500 a - 2.3 1.3 a - -

GAPDH-Sardina pilchardus a 92.0 7.4 a 73.4 8.1 a 37.6 2.9 a - -

Table 1: Kinetic parameters for oxidation reaction of GAPDH-tk

a from previously published paper in reference 8, 9, 28. - not shown.

- -
27
 6. Structure of GAPDH-tk

Base on gel filtration chromatography results, purified GAPDH-tk corresponds

to tetramer (~ 145 kDa) with a subunit molecular mass ~37 kDa. The tetrameric structure

of GAPDH-tk is not formed by disulfide bond. Most GAPDHs known to date are

homotetramers. Crysral structure of GAPDH of other organisms has been reported [25,

30]. The overall structure of eubacterial GAPDH has four identical subunits (termed O, P,

Q, and R) which are arranged with 222 point group symmetry, the 2-fold axes being

labeled P, Q, and R [30]. Each subunit composed of two domains: the NAD+ binding

domain and the catalytic domain. The NAD+ binding domain displays the characteristic

Rossmann fold, which a central parallel -sheet covered on both sides by -helices. The

catalytic domain folds into an eight-stranded antiparallel -sheet and four -helices. The

crystal structure of the apo GAPDH from Sulfolobus solfataricus (GAPDH-ss) has been

determined by multiple isomorphous replacement at 2.05 resolution [25]. GAPDH-ss

has an overall increase in the number of -helices and a relocation of the active-site

residues within the catalytic domain of GAPDH-ss. A disulphide bridge was also revealed

in a GAPDH-ss monomer between the cysteine 123 and cysteine 149, which are not

conserved in other archaeal GAPDHs. GAPDH-ss tetramer has 222 point group

symmetry and is 86 x 84 x 80 in size. The native form of rabbit GAPDH is also

composed of four identical monomers but it has no disulfide bonds in the interface and

the folded structure [27]. Although most GAPDH homologs studied thus far have a

tetrameric structure, the x-ray crystallographic structure of GAPDH from Kluyveromyces

marxianus (GAPDH-km) revealed a dimer [30]. Each dimmer of GAPDH-km has

dimensions of ~ 98 x 57x 48 .

In order to clarify the oligomeric structure of GAPDH-tk, we performed

- -
28
transmission electron microscopy (TEM) analysis. The electron micrographs of the

negatively stained GAPDH-tk showed a uniform distribution of cross-shaped structures

(fig. 11A). A total of well stained 487 particles was translationally aligned and subjected

to multivariate statistical analysis [18]. The resulting eigenimages represent all important

structural features of the original data set.

Using the 11 most significant eigenvectors, 3 classes were discriminated

according to the similarity of fearures after rotational alignment but without application

of any symmetrization. The class averages revealed a cross-shaped structure with a 2-fold

symmetry (fig. 11B). The averaged image of GAPDH-tk shows arrangement of four

subunits (fig. 11C). The arrangement of the dimers of GAPDH-tk makes the tetramer less

isometric like GAPDH-ss [25]. But the central cavity of tetramic GAPDH-tk is much

higher density than that of GAPDH-ss.

- -
29
 A

B C

Figure 11: Electron micrograph and image processing of the negatively

stained GAPDH-tk.

(A) Electron micrographs obtained by negative staining of GAPDH-tk with 2%

uranyl acetate. The arrows indicate GAPDH-tk. (B) Class averages (1-3) were derived

from the rotationally aligned images using eleven most significant eigenvectors but

without any symmetrization. (C) The correlation average of side-on views of GAPDH-tk

(487 particles).

- -
30
 7. Response of GAPDH-tk under oxidative stress.

GAPDH was considered a classical glycolytic protein examined for its pivotal

role in energy production. However, recent evidences demonstrate that GAPDH displays

a number of diverse activities unrelated to its glycolytic function [10]. GAPDH is induced

by a variety of stressors, most of which are associated with oxidative stress. Oxidative

stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the

active site cysteine, and the formation of such abnormal aggregates promotes cell death

[27]. During the past several years, GAPDHs role as a mediator for cell death has been

highlighted. A novel cell death cascade, nitric oxide-GAPDH-Siah (an E3-ubiquitin-

ligase), was revealed in eukaryotic cells [26]. Although nitric oxide mediates apoptotic

cell death, the mechanism is not well understood. The response of archaeal GAPDHs

with oxidative stress has not reported until now. So, we treated GAPDH-tk with hydrogen

peroxide (H2O2) and nitric oxide (NO) to identify the response of GAPDH-tk with

oxidative stress.

GAPDH-tk was treated either NO or H2O2 and incubated at two temperatures, 37

0
C and 80 0C, for 1 hour, 5hours and 10 hours. After treatment, the mixture was loaded on

native PAGE (fig. 12). GAPDH-tk appeared a new form with high molecular weight (~

232 kDa) when it is treated by either NO or H2O2 at 80 0C for 10 hours. However, amount

of new form treated NO increases slightly more than that treated H2O2. It indicates that

GAPDH-tk exhibits more sensitive with NO than H2O2.

Following treated condition above, these oxidized GAPDH-tk forms were

separated by using gel filtration chromatography (fig. 13). Simultaneously, the GAPDH-

tk proteins were heated at 80 0C for 10 hours without H2O2 and NO to use as negative

control. One peak was detected after heating GAPDH-tk without oxidants (fig. 13A). The

- -
31
heating GAPDH-tk has native molecular weight ~ 145 kDa with molecular mass of

subunits 37 kDa (fig. 13B). Its molecular weight is the same size with native GAPDH-tk.

This result demonstrates temperature has not effect on structure of GAPDH-tk.

Contrary to heating GAPDH-tk, two peaks were detected after H2O2 stress (fig.

13C). Oxidized GAPDH-tk proteins have two native molecular weights, ~ 232 kDa (peak

1) and 145 kDa (peak 2) (fig. 13D). However, three peaks were also detected after NO

stress (fig. 13E). Oxidized GAPDH-tk proteins from peak 1, peak 2, peak 3 have native

molecular weight ~ 669 kDa, ~ 232 kDa, ~ 145 kDa, respectively (fig. 13F). Oxidized

GAPDH-tk proteins from peak 1 and peak 2 have higher molecular weight than peak 3

under NO stress. Suggesting that oxidant induced oligomerization of GAPDH-tk.

The oxidized GAPDH-tk proteins were observed by TEM (fig. 14). In the case

of H2O2-treated GAPDH-tk, high molecular weight of GAPDH-tk (~ 232 kDa, peak 1)

appeared aggregates (fig. 14B) while low molecular weight of GAPDH-tk (~ 145 kDa,

peak2) was similar with native GAPDH-tk (fig. 14C). In the case of NO-treated GAPDH-

tk, high molecular weight of GAPDH-tk (~ 669 kDa, peak 1) formed aggregates like

short fibrils (fig. 14D) and high molecular weight of GAPDH-tk (~ 232 kDa, peak 2)

formed amorphous agglomerates (fig. 14E); but low molecular weight of GAPDH-tk (~

145 kDa, peak 3) was the same with native GAPDH-tk (fig. 14F). Taken together,

oxidative stress induced aggregation of GAPDH-tk in vitro. In addition, we checked

glycolytic activity for oxidized GAPDH-tk (fig. 16). Base on the result, oxidized GAPDH

has lost glycolytic activity.

The previous study reported that the conformational changes in the GAPDH

structure accompanied with the increase of -sheet contents were found to be critical to

the mechanism of oxidative stress-induced aggregation of rabbit GAPDH [27]. Amyloid

- -
32
fibril-like features were also detected in the resultant aggregates of GAPDH. There were

four cysteine residues in each monomer of rabbit GAPDH. They propose a model of

oxidative stress-induced oligomerization of GAPDH through formation of intermolecular

disulfide bonds. First, oxidative stress causes a conformational change of GAPDH that

allows a disulfide bond to form between Cys 149 residues of different subunits.

Subsequently, the Cys 149 Cys 149 disulfide bond induces further conformational

changes, resulting in exposure of other cysteines. The newly exposed cysteines form the

next disulfide bond, and the chain reaction accelerates formation of further multimeric

oligomers. But in the case of GAPDH-tk, GAPDH-tk aggregates were also induced by

oxidative stress although there was only one Cys 141 in each monomer. Aggregated

shapes of GAPDH-tk show various types. Aggregates of NO-treated GAPDH-tk (~ 669

kDa) were shown as fibril but it was shorter than that of aggregates of rabbit GAPDH

[27]. Additionally, amorphous agglomerates of NO-treated GAPDH-tk (~ 232 kDa) were

observed unlike aggregates of rabbit GAPDH. Oxidative stress may convert the normal

conformation of GAPDH-tk to an abnormal one. But mechanism of oxidative stress-

induced aggregation of GAPDH-tk is still unclear.

Our study reported the first description of aggregates of GAPDH from archaea

under oxidative stress. The mechanism of oxidative stress-induced aggregation of

GAPDH-tk and its new function need to examine in future works.

- -
33
 370C 800C 370C 800C

incubated time - 1h 5h 10h 1h 5h 10h 1h 5h 10h 1h 5h 10h

NO (1mM) - + + + + + + - - - - - -
H2O2 (1mM) - - - - - - - + + + + + +
M 1 2 3 4 5 6 7 8 9 10 11 12 13
669

440

232

140

67

Figure 12: Oxidized GAPDH-tk forms were visualized on 7.5 % native gel.

GAPDH-tk was incubated with oxidant (NO or H2O2) at 37 0C and 80 0C for 1

hour, 5 hours, 10 hours. The plus (+) indicates the present of oxidant, the minus (-)

indicates the absent of oxidant.

- -
34
 A 0.12 660 440 232 140 43
B

0.1
absorbance at 280nm

0.08

0.06

0.04

0.02

0
0 10 20 30 40
Time (min)

C 660 440 232 140 43

D
0.3
ab so rbance at 280n m

0.25

0.2

0.15

0.1

0.05

0
0 10 20 30 40
Time (min)

E F
0.18 660 440 232 140 43

0.16

0.14
Absobance at 280nm

0.12

0.1

0.08

0.06

0.04

0.02

0
0 10 20 30 40

Time (min)

- -
35
 Figure 13: GAPDH-tk proteins treating without or with H2O2, NO were

separated by gel filtration chromatography.

(A) Purified GAPDH-tk proteins were incubated at 80 0C for 10 hours and

loaded on Superdex 200 10/300 GL column. It was used for negative control. (B) After

heating, GAPDH-tk protein fractions from gel filtration were checked on native-PAGE

and SDS-PAGE, low panel. (C) Purified GAPDH-tk proteins were treated with H2O2 at

80 0C for 10 hours and loaded on Superdex 200 10/300 GL column. (D) After H2O2

treatment, oxidized GAPDH-tk proteins were visualized on native-PAGE and SDS-PAGE.

(E) Purified GAPDH-tk proteins were treated with NO at 80 0C for 10 hours and loaded

on Superdex 200 10/300 GL column. (F) The molecular weight of oxidized GAPDH-tk

from NO stress was determined on native-PAGE and SDS-PAGE. The stars indicate the

peak absorbance at 280 nm. The numbers indicate the fractions from gel filtration.

- -
36
 A B C

D E F

Figure 14: GAPDH-tk proteins treating without or with H2O2, NO were

observed by transmission electron microscopy.

(A) GAPDH-tk proteins were treated without H2O2 and NO at 80 0C for 10 hours.

GAPDH-tk was treated with H2O2 at 80 0C for 10 hours and separated with high

molecular weight of GAPDH-tk (~232 kDa) (B), low molecular weight (~ 145 kDa) (C).

GAPDH-tk was treated with NO at 80 0C for 10 hours and separated with high molecular

weight of GAPDH-tk (~669 kDa) (D), mediate molecular weight of GAPDH-tk (~ 232

kDa) (E), low molecular weight of GAPDH-tk (~ 145 kDa) (F).

- -
37
 11

10

8
specific activity (U/mg)

7
Native-GAPDH
6
GAPDH-H2O2-high MW
5
GAPDH-H2O2-low MW
4

0
0 1 2 3 4 5 6 7 8 9
Time (min)

11

10

8
specific activity (U/mg)

7
Native-GAPDH
6
GAPDH-NO-high MW
5
GAPDH-NO-low MW
4

0
0 1 2 3 4 5 6 7 8 9
Time (min)

Figure 15: Glycolytic activity test for oxidized GAPDH-tk forms.

Oxidized GAPDH-tk proteins from H2O2 stress (A) or NO stress (B) were
measured glycolytic activity.

- -
38
 IV. CONCLUSION

GAPDH-tk plays a role in glycolysis by catalyzing the reversible reaction of D-

glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate using NAD+ as a cofactor.

Studies on the effect of temperature and pH on enzyme activity revealed the optimal

value in the 70 0C - 90 0C interval and 7.0, respectively. GAPDH-tk exhibited high

glycolytic-activity at 80 0C and cysteine 141 is as active site of GAPDH-tk. Furthermore,

GAPDH-tk showed half-time of 5 hours at 80 0C and 90 0C.

Quaternary structure of GAPDH-tk was observed as tetramer. The tetrameric

GAPDH-tk is not formed by disulfide bonds. Nevertheless, the two distinct oligomers of

GAPDH-tk were induced by oxidative stress. Low molecular weight GAPDH-tk is

similar with native GAPDH-tk. High molecular weight GAPDH-tk formed aggregation

due to influence of oxidant. Moreover, oxidized GAPDH-tk has lost glycolytic activity.

- -
39
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 ACKNOWLEDGEMENT

I would like to express my warmest thanks and acknowledgements to all those who

encouraged me to complete my thesis.

First of all I would like to extend my heartfelt thanks to Professor Cheong Gang-Won,

my advisor, for providing me the opportunity to further develop my understanding of

biochemistry as a science. I would like to thank him for providing infinite patience,

guidance, support throughout this project and most of all for believing and trusting my

capability as a researcher, as a teacher and a leader.

I owe my special thanks to Doctor Jia Baolei for his supervision during the research

and for motivating and inspiring me over the years to broaden my interests in

biochemistry. His valuable guidance and crucial and constructive suggestions greatly

improved this study.

I would like to thank my past and present laboratory members, Lee Sangmin, Pham

Bang Phuong, Yu rui, Cho Yoon Seung, for providing invaluable knowledge, enthusiastic

help and warm friendship during my years in the Structural Biochemistry Laboratory

group.

Last but not least, I sincerely thank all my Vietnamese friends in GNU for helping me

in many ways and for sharing sorrow and happiness and making my stay in Korea

enjoyable and memorable.

Finally, I owe my loving thanks to my parents and my darling for their endless love

and support. Without their encouragement and understanding it would have been

impossible for me to finish this work.

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