人類醯亞胺水解酵素之分子選殖表現及其功能性質探討

國立交通大學

生化工程研究所

碩士論文

人類醯亞胺水解酵素之分子

選殖表現及其功能性質探討

Heterologous Expression, Purification and Functional

Characterization of Human Imidase

研究生：廖家煒

指導教授：楊裕雄 教授

人類醯亞胺水解酵素之分子選殖表現及其功能性質探討

Heterologous Expression, Purification and Functional Characterization of

Human Imidase

研 究 生：廖家煒 Student: Chia-Wei Liao

指導教授：楊裕雄 教授 Advisor: Prof. Yuh-Shyong Yang

國立交通大學

生化工程研究所

碩 士 論 文

中華民國九十七年七月

A Thesis

Submitted to Department of Biological Science and Technology

National Chiao Tung University

in Partial Fulfillment of the Requirements for the Degree of Master of

Science in Biologic Science and Technology

Hsinchu, Taiwan, Republic of China

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人類醯亞胺水解酵素之分子選殖表現及其功能性質探討

學生: 廖家煒

指導教授: 楊裕雄 教授

國立交通大學生化工程研究所碩士班

摘要

醯亞胺水解酵素(DHP, dihydropyrimidinase, EC 3.5.2.2 )參與嘧啶的還原性分 解途徑。人類醯亞胺水解酵素基因座落於染色體位置8q22，含 1560 個鹼基所構 成的開放讀碼框，編碼著由519 個胺基酸所構成的多肽鏈。人類醯亞胺水解酵素

屬 於 cyclic amidohydrolase superfamily ， 其 中 包 含 了 dihydropyrimidase 、

allantoinase、hydatoinase、dihydroorotase 等，皆參與嘌呤與嘧啶環的代謝。分析 人類醯亞胺水解酵素的胺基酸序列，其與大鼠及小鼠分別有 90%及 88%的相似 度。人類醯亞胺水解酵素缺乏症是一種體染色體隱性遺傳的疾病，不但可藉由檢 測病患尿液中dihydropyrimidine 含量來判斷是否患有醯亞胺水解酵素缺乏症，而 且與許多臨床病徵如癲癇、畸形、心理及生理發育不完全有關。然而截至目前為 止，在蛋白質層面，人類醯亞胺水解酵素仍有尚未被了解的生化特性及功能。為 了分析人類醯亞胺水解酵素的特性，我們解決在大腸桿菌內表現人類醯亞胺水解 酵素所遇到的蛋白質包涵體(Protein inclusion body)問題，在大腸桿菌內大量表現 可溶並具活性的人類醯亞胺水解酵素，並進一步比較不同物種來源，在酵素動力

學、pH 值及溫度對活性的影響、耐熱度、耐鹽度上的差異。除此之外我們還積

極地建立一個能穩定生產人類醯亞胺水解酵素的平台，以便後續能夠從蛋白質層 面來探討，在病理上如何因為單一個氨基酸突變而造成人類醯亞胺水解酵素失去 活性，導致人類醯亞胺水解酵素缺乏症的發生。

Heterologous Expression, Purification and Functional Characterization of

Human Imidase

Student: Chia-Wei Liao Advisor: Prof. Yuh-Shyong Yang Department of Biological Science and Technology and Institute of Biochemical Engineering,

National Chiao Tung University, Hsinchu, Taiwan, ROC

ABSTRACT

Imidase participates in the reductive degradation pathway of pyrimidine. The location of

Homo sapiens dihydropyrimidinase (hDHP, DPYS, NM_001385) gene is on the chromosome 8q22 and its cDNA clone contains 1560-bp open reading frames encoding polypeptides of 519 residues. hDHP belongs to the superfamily of cyclic amidohydrolase, including dihydropyrimidase, allantoinase, hydatoinase, dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings. The deduced amino acid sequence of hDHP shows 90% and 88% identity with that of rat and mouse DHP, respectively. hDHP deficiency

is an automosomal recessive disorder characterized by dihydropyrimidinuria and associated with variable clinical phenotypes, such as seizures, mental retardation, growth retardation and dysmorphic features. However, little is known about the properties and biological functions of human imidase at protein level. In order to characterize the biochemical properties of human imidase, we expressed human imidase gene in E. coli and rendered the purified protein in soluble and active form. The characterizations in enzyme kinetics, the effect of pH and temperature to enzyme activity, thermostability and salt tolerance of human imidase and those from other species were compared. This provided a basis for the future preparation of imidase mutants to study the effect of single amino acid substitution on imidase that causes hDHP deficiency

Contents Page

Abstract (Chinese)

... i

Abstract (English)

...ii

Contents

...iiii

Content of table and figure

... iv

Abbreviation and Symbol

... vi

Introduction

... 1

Experimental Procedures

... 6

Results

... 11

Discussion

... 15

Reference

... 19

Appendixe

... 40

Content of table and figure

Page

Table I Summary of purification of Nus-His-taged recombinant hDHP from E.

coli ………. 23

Table II Specific Activity of hDHP and a.r.HYD ………... 24

Table III Kinetcs constants of imidase from different sources with phathlimide … 25 Table IV Calibration curve determination of molecular weight on Sephacryl S-300 HR ……….. 26

Table V Physical and chemical parameter of hDHP ……….. 27

Figure 1 SDS-PAGE (10%) of purified recombinant hDHP ……….. 28

Figure 2 Purified recombinant hDHP analyzed by MADI-TOF after trypsin-in-gel digestion ………. 29

Figure 3 Temperature profile of hDHP ……… 30

Figure 4 pH profile of hDHP ………... 31

Figure 5 Thermostability of hDHP ……….. 32

Figure 6 Effect of urea treatment of hDHP ……….. 33

Figure 7 Effect of urea concentration of hDHP ………... 34

Figure 9 hdhp-pET-43.1a (+) construct DNA sequencing ……….. 36

Abbreviation and Symbol

Abbreviation and Symbol Full name

ε Absorption (extinction) coefficient

A280 Absorption at 280 nm

A298 Absorption at 298 nm

Bis-Tris propane 1,3-bis[ tris( hydrohxymethyl) methylamino] propane

Tris-HCl tris( hdroxymethyl) aminomethane hydrochloride

SDS dodium dodecly sulfate

PAGE polyacrylamide gel electrophoresis

hdhp dihydropyrimidinase gene of Homo sapien hDHP dihydropyrimidinase of Homo sapien

Introduction

Imidase catalyzes the hydrolytic cleavage of imide bond. Imidase is also known as

dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihiydropyrimidine hydrase, and

dihydropyrimidine amidohydrolase due to its broad substrate specificity.

Figure.1 The typical reaction catalyzed by imidase

The first research of imidase activity in living organisms was found as far back as 1926.

Gaebler and Keltch investigated the metabolism of hydantoin derivatives by detecting that the

hydantoic acid was excreted after injection of hydantoin into dogs and called this kind of

imidase as hydantoinase [1]. Afterward, many studies show that imidase activity could be

found in some tissues of different plants and animals. Up to 1957, Wallach and Grisolia isolated

imidase from calf liver and used dihydropyrimidine as nature substrate, so they called the

hydantoinase found by Gaebler and Keltch, and therefore imidase participates in the pyrimidine

metabolism [2].

The cDNA clone enconding dihydropyrimidinase from human liver had been firstly

isolated in 1996 [3]. The deduced amino acid sequence of human DHP show 90% and 88%

identity with that of rat and mouse DHP, respectively (Appendix 3). The gene of Homo sapiens

dihydropyrimidinase (hDHP, DPYS, NM_001385) locates on the chromosome 8q22

(Appendix 1) and its cDNA clone contains 1560-bp open reading frames encoding

polypeptides of 519 residues (Appendix 2) [3]. Whilecloning the hDHP cDNA, there are three DHP related protein (DRP)-1, DRP-2 and DRP-3 have been found. All of them show high identity of amino acid sequence with hDHP (Appendix 3), but without imidase activity. DHP and DRPs display differential tissue distribution, i.e. human DHP in liver and kidney; human DRP-1 in brain; human DRP-2 ubiquitously expressed except for liver; human DRP-3 mainly

in heart and skeletal muscle. The function of DRPs and their relationships with DHP in

evolution are still not clear [3].

hDHP belongs to the superfamily of cyclic amidohydrolase, including

dihydropyrimidase, allantoinase, hydatoinase, dihydroorotase, all of which are involved in

metabolism of purine and pyrimidine rings. In the biological system, pyrimidines are

metabolized by either an oxidative or a reductive pathway. It is well-known that mammals,

Dihydropyrimidinase performs reversible hydrolytic ring-opening of dihydrouracil and

dihydrothymine to N-carbamoyl-β-alanine and N-carbamoyl-β-aminoisobutyric acid,

respectively. 3-ureidopropionase catalyzes the irreversible hydrolysis of

N-carbamoyl-β-alanine and N-carbamoyl-β-aminoiosbutyric acid to β-alanine,

β-aminoisogutyric acid, ammonium ions, and carbon dioxide [4].

Figure 2. Reductive pathway of pyrimidine degradation

The reductive pyrimidine degradation pathways seem to be important especially in

mammalian cell. In mammalian tissue, it is now established that the degradation of uracil via

dihydrouracil and N-carbamoyl-β-alanine plays an important role in the synthesis of β-alanine

and β-aminoisobutyric acid. β-alanine is a structural analogue of γ-aminobutyric acid and

glycine, the important neurontransmitter in nerve system. Furthermore, β-aminoisobutyric acid

has been shown to be a potential agonist of the glycine receptor [5]. Pathologically, the defect

of dihydropyrimidine may cause the altered homeostasis of β-alanine in patients then accounted

DHP deficiency (MIM 222748) is an automosomal recessive disorder that is characterized

by dihydropyrimidinuria. To date, only nine cases of complete DHP deficiency have been

reported. These patients reveal variable clinical phenotypes, such as seizures, mental

retardation, growth retardation and dysmorphic features [6-15]. It is not a direct evidence to

infer the relationship between the homeostasis of β-alanine and the apparent clinical

abnormalities. DHP deficiency, therefore, is not only probably a necessary, but sufficient for

the onset of a clinical phenotype.

Analysis of the genotype of some patients with DHP deficiency reveals the missense at the

nucleotide location of open reading frame 1078, mutated from T to C (W360R) in exon 6 and a

novel missense mutation 1235G to T (R412M) in exon 7 [15]. Analysis of the structural effect on the DHP mutations, W360 and R412, by structure modeling indicates that W360 and R412 are located on the periphery of hDHP rather distant from the catalytic centre. This excludes a

direct effect of the amino acid exchanges on active site architecture and catalysis. Instead, the

lack of residual activity for the point mutants is likely based on global effects of the exchanges

on the protein structure [15]. However, all of these data are only based on bioinformatical

prediction without the evidence at protein level.

According to the analysis of imidase structures, all imidases are metalloproteins with

TIM-barrel architecture. All known mammalian imidases purified from the livers of bovine,

atoms, and one Zn atom per subunit. The variations are found, however, in structure, metal

content, substrate specificity and other requirement of similar enzyme from different organism.

Since then, little is known about the properties and biological functions of human imidase

at protein level. In our experience, the bottleneck is the problem of protein inclusion bodies

during expressing the hdhp gene in E. coli.In order to characterize the biochemical properties

of human imidase, we will express human imidase gene in E. coli in soluble and active form.

The differences in enzyme kinetics, the effect of pH and temperature to enzyme activity,

thermostability and salt tolerance of human imidase and those from other species will be

compared. We will also prepare imidase mutants, W360 and R412, to study the effect of single

Experimental Procedures

Materials― cDNA of hdhp gene on pCMV-sports6 vector, obtained from human gene

cDNA library in College of Biological Science and Technology, NCTU, pGEM-T

Vector(Promega), modified pET-43.1a(+) vector(Novagena) modified by Dr. LE, BL21

(DE3)pLyS competent cell(genotype: E. coli B F– dcm ompT hsdS(rB– mB–) gal λ(DE3)[pLysS

Camr](Novagen), Sephacryl S-300 HR column(amersham pharmacia biotech), High Molecular

Weight standard (code: 17-0041-01) Gel Filtration Calibration Kits(amersham pharmacia

biotech).

Molecular cloning hdhp gene into the pET-43.1a(+) vector― The PCR products of

cDNA of hdhp gene of Homo sapien on pCMV-sports6 vector was cloned into pGEM-T

Vector (Promega), and the termini of amplified DNA was added the restriction sites of BamH I

and Xho I with the pairing primers hdhp-F (the forward primer: 5'-CGGATCCATGGCGGC

GCCCTCGCG-3’) and hdhp-R (the reverse primer: 5’-CCTCGAGGGGGTG

GGCCTGTTTCCTGG-3’) by PCR. The DNA fragment containing the hdhp gene on pGEM-T

vector was subcloned into the modified pET-43.1a(+) vector by restriction enzyme, BamH I &

Xho I, and T4 DNA Ligase.

Bacterial expression, purification of hDHP― The flask culture (500 ml of LB medium

containing 50 μg/ml ampicillin) of the BL21 (DE3)pLyS cell containing the construct of hdhp

hours at 37℃ in a shaking incubator until cells reach mid-log growth (A600 of 0.8-1.0). The

culture was induced by adding 0.4mM IPTG and 1mM CoCl2, then incubated at 20℃ for

16 hours. The cells were harvested by centrifugation at 15000g for 30 min at 4℃. The cell

pellet was resuspended in 20 ml of Histrap Column Buffer A (pH 8.0) , consist of 50mM Tris,

500mM NaCl, 5mM Imidazole10% Glycerol, and the cell was lysed by sonication with 550

Sonic Dismembrantor (Fisher Scientific). The insoluble debris was removed by centrifugation

at 30000g for 30 minutes at 4℃, if necessary, repeat this step again to ensure removing the

insoluble debris completely. The sample should be filtered through a 0.45 mm filter before it

is applied to the Histrap ion exchange column(Pharmacia), equilibrated by Histrap Column

Buffer A. The column was washed with >1000 ml of Histrap Column Buffer B (pH 8.0) ,

consist of 50mM Tris, 500mM NaCl, 50mM Imidazole, 10% Glycerol, until no material

appears in the effluent. After changing the buffer in the column with Histrap Column Buffer A,

applied 40 unit of bovine thrombin to column and incubate it for >16 hours at 4 ℃ to cleave

the Nus-tag from the N-terminal of hDHP. The column was washed with 200 ml of Histrap

Column Buffer B, then The bounded protein was eluted with 50 ml of Histrap Column Buffer

C(pH 8.0), consist 50mM Tris, 500mM NaCl, 500mM Imidazole, 10% Glycerol.The product

was concentrated with Amicon Ultra-15 50K NMWL device (MILLIPORE) by centrifugation

In-gel-digestion and idetificaiton using MALDI-TOF― The spots of interest were excised

and digested in gel with trpsin according to Shevchenko’s method (Shevchenko et al., 1996).

The digested sample was taken up and analyzed using MADI-TOF. The results were

correlated with the sequence database using the NCBI and SwissProt database and analyzed

by Mascot software.

Enzyme assays― A rapid Spectrophotometric assay was used as the standard assay. the

decrease in absorbency at 298 nm was measured upon hydrolysis of phthalimide as the

substrate at 25 ℃. To start the reaction, the enzyme solution was added into 1 ml solution,

containing 1 mM phthalimide and 100 mM Bis-Tri propane at pH 7.0. Under these conditions,

a change in A298 of 2.26 represents the hydrolysis of 1 μmol of the substrate. The hydrolysis of

phthalimide was monitored with a UV/VIS spectrophotometer (Hitachi U3300).

Protein concentration determination― The protein concentration of enzyme solution

was determined by A280 or BCA protein assay (Bio-rad) using bovine serum albumin as a

standard. For the homogeneous imidase, 1 unit of A280 equals to 0.907 mg/ml imidase based

on the BCA protein assay.

Kinetic constants determination― Measurement of kinetic constant of each substrate was

performed by varying the concentration of one substrate, while keeping the other substrate at a

fixed and near saturating concentration. The apparent Km and Vmax were determined using

Temperature profile analysis― The specific activity of hDHP with phathalimide at given

temperature was measured with a UV/VIS spectrophotometer. To start the reaction, the enzyme

solution was added into 1 ml of solution containing 1 mM phthalimide and 100 mM Bis-Tri

propane to process enzyme assay at pH 7.0 and given temperaturature.

pH profile analysis― The specific activity of hDHP with phathalimide at given pH was

measured with a UV/VIS spectrophotometer. To start the reaction, the enzyme solution was

added into 1 ml of solution containing 1 mM phthalimide and 100 mM Bis-Tri propane to

process enzyme assay at given pH and 25℃

Thermostability analysis― The residual activity of hDHP with phathalimide was measured

with a UV/VIS spectrophotometer. Prior to start the reaction, the enzyme solution was

incubated in 1 ml of 100 mM Bis-Tri propane(pH 7.0) for time course at given temperature,

then added 1mM phtalimide to process enzyme assay.

Salt tolerance analysis―The residual activity of hDHP with phathalimide was measured

with a UV/VIS spectrophotometer. Prior to start the reaction, the enzyme solution was

incubated in 1 ml of the given concentration urea and 100 mM Bis-Tri propane(pH 7) for time

course at 25℃, then added 1mM phtalimide to process enzyme assay.

Size-exlusion chromatography and calibration curve preparation― Aliquots of 1 ml of

various samples were applied on a Sephacryl S-300 HR column that was equilibrated with 20

flow rate of 1.0 ml/min. Molecular weights were estimated against Gel Filtration Calibration

Kits(amersham pharmacia biotech) consisted of aldolase(178 KDa), catalase(228 KDa), ferritin

(437 KDa), thyroglobulin(692 KDa), Blue dextran(2000 KDa). Molecular weight of hDHP was

Result

Molecular Cloning of hdhp gene into the pET-43.1a(+) vector― we constructed hdhp gene on different vectors to screen an appropriated vector (Fig. 9, only show the hdhp-pET-43.1a (+) construct) to express soluble and active hDHP. We found pET-43.1a (+)

vector can overexpress the soluble and active hDHP.

Bacterial Expression and Purification of hDHP―The pET-43.1a(+) vector provides the hDHP for fusion to Nus-tag and His-tag on N-terminus and C-terminus, respectively. The

Nus-His-taged fusion protein was expressed in the BL21 (DE3)pLyS cell and purified by

Histrap column as describe in “Experimental Procedure”. The enzyme was finally purified

about 343.7-fold with 49% yield (Table I) and in a homogenous form according to the

identifications of SDS-PAGE (Fig. 1) and MADI-TOF data (Fig. 2). These data display that we

are the first one to not only purify hDHP in a homogenous and active form but also express the

mammalian imidase in heterolous system. We analyzed the biochemical characterization to

deduce the difference in enzyme kinetics, the effect of pH and temperature to enzyme activity,

thermostability and salt tolerance of human imidase between from human and other species.

Substrate specificity and kinetic constants of the cobalt-amended hDHP with different substrates― A number of xenobiotic and physiological substrates were tested as substrates for hDHP (Table II). The specific activities of the different substrates of hDHP were determined

concentration of substrates are 1 mM. The specific activities of hDHP with dihydrouracil,

hydantoin, phthalimide, and maleimide are 0.37, 3.48, 24.49, and 29.72 μmol/min/mg,

respectively. hDHP has higher specific activities with phthalimide, and maleimide in the

condition. The kinetic constants of hDHP with phathalimide were determined experimentally

by direct measurement with a spectrophotometer as indicated under “Experimental

Procedures”. Km, kcat, and kcat/Km of of hDHP with phathalimide are 0.65, 2615, and 4021

μmol/min/mg, respectively (Fig. 10).

The temperature and pH profile of hDHP― The temperature profile of hDHP is displayed

by the specific activity of hDHP with phathalimide at given temperature measured with a

UV/VIS spectrophotometer as indicated under “Experimental Procedure” (Fig. 3). The specific

activities of hDHP with phathalimide from 20 ℃ to 35 ℃ are all about 24.0 μmol/min/mg

without significant different. The pH profile of hDHP is displayed by the specific activity of

hDHP with phathalimide at given pH measured with a UV/VIS spectrophotometer as indicated

under “Experimental Procedure” (Fig. 4). The specific activities of hDHP with phathalimide at

pH6.5, pH7.0~7.5, 8.0~9.0, and 9.5 are about 14.2, 25.4, 31.2, and 24.5 μmol/min/mg,

respectively. hDHP has higher specific activity at pH 8.0~9.0.

The thermostatbility of hDHP― The thermal stability of hDHP is displayed by the

residual activity of hDHP with phathalimide measured with a UV/VIS spectrophotometer as

(Fig. 5). The residual activies of hDHP with phathalimide after treating at 30 ℃ for 60, 120,

and 150 minutes are 98.53, 96.19, and 95.42 %, respectively. The residual activies of hDHP

with phathalimide after treating at 37 ℃ for 60, 120, and 150 minutes are 71.54, 48.53, and

45.67 %, respectively. The residual activies of hDHP with phathalimide after treating at 50 ℃

for 60, 120, and 150 minutes are 15.66, 6.57, and 2.19 %, respectively. The residual activity

of hDHP with phathalimide after treating at 60 ℃ for 15 minutes is 1.30 %. hDHP is much

unstable above 37℃.

The effect of urea treatment of hDHP― The effect of urea treatment of hDHP is displayed

by the residual activity of hDHP with phathalimide and measured with a UV/VIS

spectrophotometer as indicated under “Experimental Procedure” after treating in given

concentration of urea for time course (Fig. 6). The residual activity of hDHP with 0.1M, 1M,

2M, 3M, and 4M urea after treating for 15minutes are 70.01, 57.33, 25.87, 9.01, and 2.39%,

respectively. The residual activity of hDHP with 0.1M, 1M, 2M, 3M, and 4M urea after

treating for 30 minutes are 67.97, 49.34, 21.18, 8.76, and 0.71, respectively. After treating in

different concentration of urea for 30 minutes, the residual activity of hDHP performed stable

and didn’t change.

Analysis of the quaternary structure of hDHP by gel filtration― The subunit size and native

size of hDHP was estimated by its deduced amino acid sequence (A. 6) and gel filtration(Table

retention volume of the blue dextran 2000 was 37.28 ml and the Kav could be estimated by the

equation: Kav= (Ve-Vo)/(Vt-Vo). The calibration curve was plotted as the independent variable

was log(M.W.) versus the dependent variable Kav as show in Fig. 8. The retention volume of

hDHP and a.r.HYD were 54.31 ml and 60.40 ml, respectively (Table V). The corresponding

molecular weight according to the retention time was shown in Fig. 8 and the calculated values

were approximately equal to the theoretical molecular weight of hDHP and a.r.HYD (Table V).

Discussion

Since then, little is known about the properties and biological functions of human imidase

at protein level, in our experience, the bottleneck is the problem of protein inclusion bodies

during expressing the hdhp gene in E. coli. Here, the protein inclusion bodies mean the

cytoplasmic aggregates of misfolded protein. This phenomenon was often founded during

expressing the eukaryotic gene in heterologous system, especially in E. coli, and it was due to

the internal microenvironment of E. coli may differ from that original source of the gene. A

variety of methods have been published describing solving the problem of protein inclusion

bodies. The choice of vector and expression host can significantly increase the activity and

amount of target protein present in the soluble fraction. An appropriate vector can enhance

solubility and/or folding by providing for fusion to a polypeptide that itself is highly soluble

(e.g.,NusA , GST, Trx), or providing for fusion to an enzyme that catalyzes disulfide bond

formation (e.g., thioredoxin, DsbA, DsbC), or provide a signal sequence for translocation into

the periplasmic space (pelB, DsbA, DsbC). In this study, we expressed the hdhp gene on the

pET-43.1a(+) vector which provided for fusion to NusA (A. 4,5). Nus A, N utilization

substance A, is the transcription factor of E. coli naturally. Nus A not only is high soluble but

also can increase the solubility of the full-length fusion protein to solve the problem of protein

inclusion bodies during expressing the hdhp gene in E. coli.

Agrobacterium [22], Blastobacter [23], Arthrobacter [24], bovine liver[16], calf liver[17], pig

liver[18,25], and rat liver[11,19], and most purifications of these were conducted by three or

four chromatography steps. We solved the problem of protein inclusion bodies during

expressing the hdhp gene in E. coli and developed a time-and-effort-saving, inexpensive, high

quality and quantity platform to express and purify homogenous hDHP with only one

chromatography step for further biochemical characterization to deduce the difference of

specific activity and structure of imidase between human and other species.

A number of xenobiotic and physiological compounds were tested as substrates for

enzymes, and the specific activities of hDHP were compared with a.r.HYD (hydantoinase from

Agrobacterium radiobacter NRRL B1) referred to the thesis of Yi-Rong Chen (Table II)[25].

The catalytic efficiencies of hDHP with the substrates are higher than that of a.r.HYD except

dihydrouracil, the six-member ring substrate. The kinetic constants, Km, Kcat, and Kcat/Km,

of hDHP with phathalimide as substrate are 0.65mM, 2615min-1, and 4021mM-1min-1,

respectively. Comparing with imidases from fish liver, pig liver, and a.r.HYD, hDHP performs

better substrate affinity and catalytic efficiency (Table III).

The temperature profile of hDHP is displayed by the specific activity of hDHP with

phathalimide at given temperature (Fig. 3). hDHP performs stable catalytic efficiency from 20

℃ to 35 ℃. It is to deserve to be mentioned the limit of enzyme assay that the substrate is

we can not measure the initial rate of hDHP activity with phathalimide at the high temperature

and the specific activity at 40 ℃ is measured much lower than which at 35 ℃. The pH profile

of hDHP is displayed by the specific activity of hDHP with phathalimide at given pH

measured (Fig. 4). hDHP performs stable catalytic efficiency from pH 8 to pH 9.

The thermal stability of hDHP is displayed by the residual activity of hDHP with

phathalimide after treating at given temperature for time course (Fig. 5). hDHP performs stable

catalytic efficiency, the residual activity is 95.4% after treating at 30 ℃ for 150 minutes. The

residual activities of hDHP are 45.8% and 2.2% after treating at 37 ℃ and 50 ℃ for 150

minutes, respectively. After treating at 60 ℃ for 15 minutes, the residual activity of hDHP is

only 1.3%. Under the same treatment, 50 ℃ for 1 hour, the residual activities of hDHP,

a.r.HYD, and fish imidase are about 13.6%, 93%, and 90%, respectively. So, hDHP is more

thermolabile than a.r.HYD and fish imidase.

The effect of urea treatment of hDHP is displayed by the residual activity of hDHP with

phathalimide after treating in given concentration of urea for time course (Fig. 6). After treating

in different concentration of urea for 30 minutes, the residual activity of hDHP performed

stable and didn’t change, so we can simply determine the stability of urea treatment of hDHP by

measuring [Urea]50%, the concentration of urea at 50% residual activity of hDHP treated for 30

R412, with wild hDHP to study the effect of single amino acid substitution on hDHP that cause

human DHP deficiency. The [Urea]50% of wild hDHP is about 1 M.

The subunit size and native size of hDHP was estimated by its deduced amino acid

sequence (A. 6) and gel filtration (Table V). This data indicated that the native form of hDHP is

homopentameric different from other mamammalian imidases, such as calf [17], pig (5) rat [19],

yeast and slime mold DHPs [25] are homotetrameric.

In this study, it is the first one solved the problem of protein inclusion bodies during

expressing the hdhp gene in E. coli, then developed a time-and-effort-saving, inexpensive, high

quality and quantity platform to express and purify homogenous soluble and active hDHP with

only one chromatography step. We have analyzed the complete biochemical characterization of

hDHP. These results indicate that hDHP performs very unique biochemical characterization on

specific activity, thermal stability, and the quaternary structure compared with the imidases

from other species. It is worthy to know which reason caused it, maybe the sequence or

structure, and its significance on evolution. We will resolve and clarify that in the future. On the

other hand, we will prepare imidase mutants, W360 and R412, then analyzed the difference of

the structure stability and other biochemical characterization of them to study the effect of

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Table I

Summary of purification of Nus-His-taged recombinant hDHP from E. coli.

a_{.“Histrap Elute After Thombin Digest” represented the step of purification of hDHP with Histrap ion exchange column as described in}

Table II

Specific Activity of hDHP and a.r.HYD

29.72±0.54 4.7±0.1 Maleimide 24.49±0.50 0.76±0.00 Phthalimide 3.48±0.03 0.18±0.01 Hydantoin 0.37±0.00 5.08±0.06 Dihydrouracil hDHP a.r.HYDa Specific Activity(μmol/min/mg) b Substrate 29.72±0.54 4.7±0.1 Maleimide 24.49±0.50 0.76±0.00 Phthalimide 3.48±0.03 0.18±0.01 Hydantoin 0.37±0.00 5.08±0.06 Dihydrouracil hDHP a.r.HYDa Specific Activity(μmol/min/mg) b Substrate

a. _{a.r.HYD represented Agrobacterium radiobacter hydantoinase. The specific activities of the}

different substrates of a.r.HYD were referred to the thesis of Yi-Rong Chen.

b. _{The specific activities of the different substrates of hDHP were determined with UV/VIS}

spectrophotometer as indicated under “Experimental Procedure”, and all the concentration.of

Table III

Kinetc constants of imidase from different sources with phathlimide

41 33 ± 1 0.8 ± 0.04 Pig liver 790 1270 ± 80 1.6 ± 0.1 Fish liver 10 40 ± 15 3.8 ± 1.8 a.r.HYD 4021 2615 ± 75 0.65 ± 0.07 hDHP kcat/ km(mM-1 min -1) kcat(min -1) km(mM) 41 33 ± 1 0.8 ± 0.04 Pig liver 790 1270 ± 80 1.6 ± 0.1 Fish liver 10 40 ± 15 3.8 ± 1.8 a.r.HYD 4021 2615 ± 75 0.65 ± 0.07 hDHP kcat/ km(mM-1 min -1) kcat(min -1) km(mM)

a. _{a.r.HYD represented Agrobacterium radiobacter hydantoinase. The kinetc constants of}

imidases from fish (Oreochromis niloticus) liver, pig liver, and were a.r.HYD referred to

the thesis of Cheng-Yang Huang

b._{The kinetic constants of hDHP with phathalimide as substrate were determined with UV/VIS}

Table IV

Calibration curve determination of molecular weight on Sephacryl S-300 HR

0.03 40.06 2.84 692.00 Thyroglobulin 0.15 49.58 2.64 437.00 Ferritin 0.26 59.06 2.36 228.00 Catalase 0.28 60.36 2.25 178.00 Aldolase Kavb Vea log(MW) MW 0.03 40.06 2.84 692.00 Thyroglobulin 0.15 49.58 2.64 437.00 Ferritin 0.26 59.06 2.36 228.00 Catalase 0.28 60.36 2.25 178.00 Aldolase Kavb Vea log(MW) MW a. _V

e represente the retention volume of eluted protein under the condition as indicated under

“Experimental Procedures”.

b. _K

av was calculated by the equation: Kav = (Ve-V0)/(Vt-V0). The Vt was the column volume as

120 ml and the V0 was the retention volume of blue dextran 2000 (2000 KDa), whose

Table V

Physical and chemical parameters of hDHP

60.40 5.46 49939 a.r.Hydantoinase 54.31 6.58 59439 hDHP V_eb Theoretical pIa Molecular weight a 60.40 5.46 49939 a.r.Hydantoinase 54.31 6.58 59439 hDHP V_eb Theoretical pIa Molecular weight a

a. _{The molecular weight and theoretical isoeletric point(pI) were determined by}

the ProtParam on ExPASy server (http://tw.expasy.org/tools/protparam.html).

b. _{The Ve indicated the retention volume as described above and determined by}

Figure 1. SDS-PAGE (10%) of purified recombinant hDHP. Lane 1, pellet; lane 2,

supernatant; lane 3, Histrap elute after thrombin on-column digest. The steps of purification of

Figure 2. Purified recombinant hDHP analyzed by MALDI-TOF after trypsin-in-gel digestion. The purified recombinant hDHP on SDS-PAGE were excised and digested in gel

with trpsin according to Shevchenko’s method (Shevchenko et al., 1996). The digested samle

was taken up and analyzed using MADI-TOF. The results were correlated with the sequence

Temperature Profile of hDHP 0 5 10 15 20 25 30 10 15 20 25 30 35 40 45 Temperature (℃ ) Spe ci fi c A cti vi ty (μ mo l/ m in /m g

Figure 3. Temperature profile of hDHP. The temperature profile of hDHP is displayed by the

specific activity of hDHP with phathalimide at given temperature measured with a UV/VIS

pH Profile of hDHP

0 5 10 15 20 25 30 35 6 6.5 7 7.5 8 8.5 9 9.5 10 pH S p ec if ic A ct iv it y ( μ mol /mi n /mg)

Figure 4. The pH profile of hDHP. The pH profile of hDHP is displayed by the specific activity of

hDHP with phathalimide at given pH measured with a UV/VIS spectrophotometer as indicated under

hDHP Thermostability 0 10 20 30 40 50 60 70 80 90 100 0 15 30 45 60 75 90 105 120 Time (min) R es id u al A cti vi ty (%) 30℃ 37℃ 50℃ 60℃

Figure 5. Thermal stability of hDHP. The thermal stability of hDHP is displayed by the

residual activity of hDHP with phathalimide measured with a UV/VIS spectrophotometer as

Effect of Urea Treatment 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Time (min) R es id u al A cti vi ty (% ) 0.1M 1M 2M 3M 4M

Figure 6. Effect of urea treatment of hDHP. The effect of urea treatment of hDHP is

displayed by the residual activity of hDHP with phathalimide measured with a UV/VIS

spectrophotometer as indicated under “Experimental Procedure” after treating in given

Urea-induced fractional unfolding of hDHP 0 10 20 30 40 50 60 70 80 90 100 0 0.5 1 1.5 2 2.5 3 3.5 4 Urea Concentration (M) R es id ua l A cti vi ty (% )

Figure 7. Urea-induced fractional unfolding of hDHP. Determine the stability of urea

treatment of hDHP by measuring [Urea]50%, the concentration of urea at 50% residual activity

Figure 8. Calibration curve of Sephacryl S-300 HR. A molecular weight calibration curve,

which defined the relationship between the elution volumes of a set standards and the logarithm

of their respective molecular weights, was determined with Sephacyl S-200 HR. Calibration

standard(crisscross) used were High Molecular Weight standard of Gel Filtration Calibration

Kit, such as aldolase (178 KDa), catalase (228 KDa), ferritin (437 KDa), thyroglobulin (692

KDa), blue dextran (2000 KDa). The hDHP and a.r.HYD were closed and open diamonds,

Figure 9. hdhp-pET-43.1a (+) construct DNA sequencing. The DNA sequence of

hdhp-pET-43.1a (+) construct was analyzed according to Senger Method by MISSION

Michaelis-Menten

Phthalimide (mM) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Activity ( umol/min /mg ) 0 5 10 15 20 25 30

Figure 10. Enzyme Kinetics of hDHP with Phthalimide. Measurement of kinetic constant

of each substrate was performed by varying the concentration of one substrate, while keeping

the other substrate at a fixed and near saturating concentration. The apparent Km and Vmax

were determined using nonlinear regression by Sigmaplot 2001,V7.0 and Enzyme Kinetics

Module, V1.1.

Km= 0.66mM

Vmax= 44.3 umol/min/mg Km= 0.66mM

Appendix

Appendix 1. The structure organization and chromosome localization of hdhp gene. (http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000147647)

Appendix 2. Nucleotide sequence of cDNA and deduced amino acid sequence of hdhp gene. Analyze the nucleotide sequence of cDNA and deduced amino acid sequence of hdhp

Appendix 3. The identity of the amino acid sequences of imidase from other species and Homo Sapien. Analyze the the amino acid sequences of imidase from other species and Homo Sapien by ClustalW2( http://www.ebi.ac.uk/Tools/clustalw2/index.html

Appendix 5. The map of hdhp-pET-43.1a (+) construct. Clone the PCR products of cDNA of

hdhp gene of Homo sapien on pCMV-sports6 vecto into pGEM-T Vector (Promega) and add

restriction sites of BamH I and Xho I to the termini of amplified DNA with the pairing primers hdhp-F and hdhp-R . Subclone the DNA fragment containing the hdhp gene on pGEM-T vector into the modified pET-43.1a(+) vector by restriction enzyme, BamH I & Xho I, and T4 DNA Ligase.

Appendix 6. Physico-chemical parameters of hDHP. Analyze the Physico-chemical

parameters of hDHP by ProtParam tool on the ExPASy sever

Appendix 7. Multiple amino acid sequences alignment of imidase from other species and Homo Sapien. Analyze the Multiple amino acid sequences alignment of imidase from other species and hDHP by ClustalW2 ( http://www.ebi.ac.uk/Tools/clustalw2/index.html). The labeled amino acids, such as His67, His 69, Lys159, His192, His 248, and Asp 326 are responsible for metal binding.