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Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme

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Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate

synthase from Thermotoga maritima: how is it different from that

of the mesophilic enzyme

Tun-Hsun Kuo

a

, Po-Huang Liang

a,b,

*

a

Institute of Biochemical Sciences, National Taiwan University, Taipei 10098, Taiwan

b

Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan Received 11 April 2002; received in revised form 2 July 2002; accepted 1 August 2002

Abstract

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s 1but product release limits the steady-state rate at 0.02 s 1[Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 jC was 0.005 s 1under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C40and the rate of its conversion to C45was negligible. Under single-turnover condition with 10 AM OPPsFPP complex and 1 AM IPP, only the C20was formed rather than C20– C40observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 jC, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Prenyltransferase; Ubiquinone; Single-turnover; Thermophile; Mesophile

1. Introduction

Thermophiles are organisms that can grow under high temperature conditions ( > 50 jC). Proteins in thermophiles are heat resistant and maintain proper three-dimensional structure even at extremely high temperatures, allowing the organism to survive the harsh environment. However,

little is known about how the proteins of a thermophile differ from those of a mesophile to account for their thermostability. Sequence comparisons show that hyper-thermophilic and mesophilic versions of the same enzyme typically share about 30 – 50% identity[1]. Even the crystal structure for a hyperthermophilic rubredoxin containing only 53 amino acids from the archaebacterium Pyrococcus furiosus shows that it is virtually superimposable on its mesophilic counterpart[2]. The relatively minor changes in protein structure apparently enhance packing via additional interactions such as salt bridges and hydrogen bonds.

In this study, a putative octaprenyl pyrophosphate syn-thase (OPPs) identified from Thermotoga maritima genome by sequence comparison (Fig. 1)was chosen as the model system to examine the activity, product specificity, thermal stability and structural flexibility of the enzyme in compar-ison with its mesophilic counterpart from Escherichia coli. T. maritima was originally isolated from geothermal heated marine sediment at Vulcano, Italy. This organism is an

1570-9639/02/$ - see front matterD 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 4 1 0 - 7

Abbreviations: OPPs, octaprenyl pyrophosphate synthase; OPP, octap-renyl pyrophosphate; FPP, farnesyl pyrophosphate; IPP, isopentenyl pyrophosphate; PCR, polymerase chain reaction; IPTG, isopropyl-h-D -thiogalactopyranoside; Ni-NTA, nickel nitrilo-tri-acetic acid; Tris, tris(hy-droxymethyl)aminomethane; Hepes, 4-(2-hydroxyethyl)-1-piperazineetha-nesulfonic acid; EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin layer chromatography; MW, molecular weight

*

Corresponding author. Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan. Tel.: +886-2-2785-5696x6070; fax: +886-2-2788-9759.

E-mail address: phliang@gate.sinica.edu.tw (P.-H. Liang).

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anaerobic bacterium and has optimum growth temperature of 80 jC[3]. OPPs belongs to a prenyltransferase family that catalyzes the chain elongation of allylic pyrophosphate via condensation with IPP [4,5]. As shown in Scheme 1, the enzyme catalyzes the condensation reactions of farnesyl pyrophosphate (FPP) with five molecules of isopentenyl pyrophosphate (IPP) to produce C40 octaprenyl

pyrophos-phate (OPP)[6,7]. OPPs is responsible for the biosynthesis of ubiquinone side chain in E. coli [8,9]. Other organisms contain ubiquinone with designate lengths of side chain synthesized by specific prenyltrasnferases[10]. For example, the lengths of ubiquinone side chain are C30 for yeast S.

cerevisiae, C45 for rat and C50 for human, which are

synthesized by hexaprenyl pyrophosphate synthase, solane-syl pyrophosphate synthase and decaprenyl pyrophosphate

synthase, respectively[11 – 13]. We had studied the OPPs in E. coli and found that the enzyme generated products longer than C40 [14]. However, the rate constant (0.02 s

1

) for formation of the product with additional IPP condensation is 100-fold smaller than that (2 s 1) for C40production.

More-over, under the condition with E. coli OPPsFPP (10 AM) in much excess of IPP (1 AM), C20– C40rather than C20alone

were generated[14]. This may be due to a higher IPP affinity of OPPsintermediate relative to OPPsFPP, a property reflecting the flexible protein conformation[15]. We report in this paper the characterization of a recombinant OPPs from T. maritima focusing on its reaction kinetics, product specificity, thermal stability and structural flexibility.

2. Materials and methods 2.1. Chemicals

[14C]IPP (55 mCi/mmol) radiolabeled substrate was purchased from Amersham Pharmacia Biotech and FPP was product of Sigma Co. Reverse phase TLC for product analysis was obtained from Merck (Darmstadt, Germany). The plasmid mini-prep kit, DNA gel extraction kit and Ni-NTA were the products of QIAGEN. Potato acidic phos-phatase (2 Unit/mg) was purchased from Boehringer Man-nheim. The pET-32Xa/LIC vector, competent cells E. coli JM109 and BL21 (DE3), T4 DNA polymerase, and Factor Xa were obtained from Novagen. All other buffer and reagents were of the highest commercial purity. Millipore ultrapure H2O was used in all experiments.

2.2. Overexpression of OPPs from T. maritima

T. maritima obtained from American type culture collec-tion (ATCC) was grown at 80 jC under anaerobic

con-Fig. 1. Alignment of T. maritima OPPs (TC40) sequence with that of E. coli OPPs (C40), Synechocystis sp. solanesyl pyrophosphate synthase (C45) and M. tuberculosis heptaprenyl pyrophosphate synthase (C35). Black and gray outlines indicate identical and similar amino acid residues, respectively.

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dition in the medium prepared according to protocol provided by ATCC. The genomic DNA was obtained from the harvested cells using DNA extraction kit according to manufacturer’s instruction. Using its genomic DNAs as template, the gene encoding OPPs from the bacterium was amplified by carrying out polymerase chain reaction (PCR). The forward primer 5V ATGACGAAAAACA-AGCTGAACCAA 3V and reverse primer 5V TCATGAA-GAGATTTTGATTTTAAA 3V were utilized in the PCR. The PCR product (OPPs gene) was purified from 0.8% agarose gel electrophoresis and used as a template for the second PCR to create FXa cleavage site and the comple-mentary sequences for the vector pET-32Xa/LIC. In this PCR reaction, the forward primer 5V GGTATTGAGG-GTCGCATGACGAAAAACAAG 3Vand the reverse primer 5V AGAGGAGAGTTAGAGCCTCATGAAGAGATT 3V were employed. The DNA product was ligation with the vector and transformed into E. coli BL21 (DE-3) for protein expression as previously described [14].

2.3. Purification of recombinant T. maritima OPPs The purification procedure of recombinant T. maritima OPPs was the same as previously reported for E. coli OPPs [14]. The cell lysate was loaded onto a Ni-NTA column and the protein with His tag was finally eluted with 25 mM Tris (pH 7.5), 150 mM NaCl and 300 mM imidazole. The protein solution was dialyzed twice against 2 l buffer (25 mM Tris, pH 7.5, and 150 mM NaCl) and digested with FXa to remove tag. The untagged protein was separated from the tag by loading onto a Ni-NTA column. The tag was bound to Ni-NTA and the OPPs eluted by a buffer of 25 mM Tris, pH 7.5, 5 mM imidazole and 150 mM NaCl was highly pure according to SDS-PAGE analysis.

2.4. Steady-state Kmand kcatmeasurements

The OPPs reaction was initiated by adding 0.1 AM enzyme (final concentration) to a mixture containing various concentrations of FPP (1 – 10 AM) and [14C]IPP (1 – 50 AM) in 100 mM Hepes buffer (pH 7.5), 50 mM KCl and 0.5 mM MgCl2 at 25 jC. The enzyme

concen-tration used in all experiments was determined from its absorbance at 280 nm (extinction coefficient=20340 M 1 cm 1). Within 10% substrate depletion, the reaction mixture was periodically withdrawn. The reaction was terminated by adding 10 mM (final concentration) EDTA and the product was extracted with 1-butanol. The prod-uct was quantitated by counting the radioactivity in butanol phase ([14C]IPP was in aqueous phase) using a Beckmann LS6500 scintillation counter. The OPPs steady-state kcat was calculated based on the rate of IPP

consumption. The initial rate was calculated by plotting the [IPP] consumed versus time and the kinetic constants were obtained by fitting the data with the Michaelis –

Menten equation using KaleidaGraph computer software (synergy software).

2.5. Temperature dependence of OPPs activity

The activity was measured as described above in a reaction mixture containing 0.1 AM OPPs, 5 AM FPP, 50 AM [14C]IPP in a buffer of 100 mM Hepes (pH 7.5), 0.5 mM MgCl2 and 50 mM KCl at temperature ranging

from 25 to 85 jC. The enzyme was added to a pre-heated mixture to initiate the reaction. The initial rate in the first 5 min of reaction was measured at each temper-ature.

2.6. Single-turnover experiments

The single-turnover reaction was initiated by mixing 15 Al of the enzyme (10 AM) preincubated with FPP (2 AM) with equal volume of [14C]IPP (50 AM) solution in buffer containing 100 mM Hepes (pH 7.5), 0.5 mM MgCl2and

50 mM KCl at 25 jC. The concentrations cited in the parentheses and hereafter in the paper are those after mixing. The reaction mixture quenched with EDTA in specified time period was extracted with the same volume of 1-butanol and the radioactivity in the organic phase (intermediates and product) was counted by the scintilla-tion counter (Beckmann LS6500). For identificascintilla-tion of intermediates and product at each time point, the radio-labeled polyprenyl pyrophosphates were extracted with 1-butanol and treated with 20% propanol, 4.4 U/ml acidic phosphatase, 0.1% Triton X-100 and 50 mM sodium acetate (pH 4.7) to be converted to the corresponding polyprenols [16]. The products were separated by reverse phase TLC with acetone/water (19:1) as mobile phase and the product distribution was determined by autoradiogra-phy [17]. The time course for each intermediate was simulated by KINSIM computer program as described below.

2.7. Data simulation

The KINSIM program[18]simulation of the kinetic data presented in this paper is as described in our previous report

[14]. The derived kinetic pathway of the T. maritima OPPs reaction is shown inScheme 1and the details of the kinetic simulation of the data are shown in Chart 1.

2.8. OPPs molecular weight determination

The molecular weight of the recombinant OPPs was determined by size-exclusion chromatography on a Blue Dextran 2000 calibrated pre-packed Sephadex G-200 col-umn (120 cm, Amsheram Pharmacia Biotech). The molecular weight was estimated from the plot of Kav vs.

log MW of protein molecular weight standards, which were catalase (206,000), aldolase (170,000), bovine serum

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albumin (67,000), and ovalbumin (43,000). A buffer con-taining 25 mM Tris (pH 7.5) and 150 mM NaCl was used to elute the proteins at a flow rate of 0.5 ml/min. The Kav

values were calculated using the equation Kav=(Ve Vo)/

(Vt Vo) where Veis elution volume of the protein, Vois the

elution volume of Blue Dextran 2000 and Vt is total gel

bed volume [19].

2.9. Circular dichroism (CD) experiments

For determination of secondary structure of T. maritima and E. coli OPPs enzymes, CD measurements were made on a Jasco J-710 spectrophotometer in a 0.1 cm water-jacketed cuvette. The ellipticity of 200-Al sample containing 1 AM OPPs in a buffer of 25 mM Tris (pH 7.5) and 150 mM NaF 25 jC was recorded from 200 to 260 nm. Spectra reported were the average of three scans collected at 30 nm/min with a 2-s response time. The secondary structure of the protein was analyzed using the program SELCON2 [20]. For the measurement of thermal stability, the ellipticity of 10 AM enzyme was monitored at 208 nm and the temperature was increased at a rate of 30 jC/h. Thermal unfolding curve of E. coli OPPs was fitted with a two-state unfolding model

[21].

2.10. Final product distribution

The reaction mixture containing different concentrations of OPPs, [14C]IPP and FPP in buffer of 100 mM Hepes buffer (pH 7.5), 0.5 mM MgCl2 and 50 mM KCl, was

incubated at 25 jC for 100 h. The reaction was terminated with 10 mM EDTA. We then extracted the products with 1-butanol, evaporated the solvent under N2, converted the

products to polyprenols and analyzed the polyprenols by TLC as described above.

3. Results

3.1. Temperature dependence of the T. maritima OPPs activity and the Tmvalue

The kcat value of recombinant OPPs of T. maritima is

0.005 s 1 and FPP and IPP Kmvalues are 1.5 and 2 AM,

respectively, at 25 jC and pH 7.5. The enzyme activities at temperatures ranging from 25 to 85 jC exponentially increased with elevated temperature (Fig. 2A). According to Arrhenius equation, the activation energy (Ea) required

for the reaction is calculated to be 16.2 kcal/mol. Measured by CD spectrophotometer, the temperature at which T. maritima OPPs is half-folded (Tm) is >80 jC, higher than

that of E. coli enzyme (55.3 jC), indicating that T. maritima OPPs has higher thermal stability (Fig. 2B).

Plate 1. The intermediates and product formed during the T. maritima OPPs single-turnover reaction with 10 mM enzyme, 2 mM FPP and 50 mM [14C]IPP at pH 7.5 and 25 jC. The reaction continued for 120 to 960 s and an extended period of 2400 s.

Fig. 2. (A) T. maritima OPPs activity measured at different temperature. The initial rate in the first 5 min of the enzyme reaction of 0.1 AM enzyme with 5 AM FPP and 50 AM [14C]IPP was measured at temperature ranging from 25 to 85 jC. (B) Thermal stabilities of recombinant OPPs from E. coli and T. maritima monitored by CD. The ellipticity at 208 nm was monitored from 25 to 90 jC using 10 AM E. coli OPPs (D) and 10 AM T. maritima enzyme (

.

) The half-folded Tmwas 55.3 jC for E. coli enzyme by fitting

the data with a two-state unfolding model. For T. maritima OPPs, the unfolding was not completed at 90 jC.

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3.2. Complete kinetic pathway of T. maritima OPPs Due to the great hydrophobicity of the product, the reaction rates of the long-chain polyprenyl synthetic enzymes are often limited by slow product release under steady-state condition [14,22,23]. The 3-D structure of the C55-undecaprenyl pyrophosphate synthase implicates the

strong hydrophobic interactions between the enzyme and the product and provides a rationale for the slow product release[24]. In order to measure the IPP condensation rate constant, we have to perform the single-turnover experi-ments with higher enzyme concentration than the substrate

FPP so that product release is not limiting the reaction rate

[14,22,23]. The formation of C20– C40 catalyzed by T.

maritima OPPs was examined during the enzyme single-turnover reaction containing 10 AM enzyme, 2 AM FPP and 50 AM [14C]IPP. The total formation of radiolabeled species with time in the reaction is shown in Fig. 3. Since the percentages of C20– C40 at each time point of the reaction

were obtained by imaging as shown in Plate 1, the time courses of these intermediates could be determined(Fig. 4). The data were fitted using KINSIM program to obtain rate constant for individual IPP condensation catalyzed by OPPs. The derived kinetic pathway of T. maritima OPPs reaction is summarized in Scheme 1. The fit of the total IPP

incorpo-Fig. 3. The single-turnover reaction of T. maritima OPPs with enzyme in excess of FPP. A solution containing enzyme (10 AM) preincubated with FPP (2 AM) was mixed with [14C]IPP (50 AM) at pH 7.5 and 25 jC. The

products of IPP condensation formed at each time point were quantitated by scintillation counting of the radioactivity in the butanol layer. The curve represents a fit by KINSIM simulation using rate constants shown in

Scheme 1.

Fig. 4. Single-turnover time courses of intermediates (C20– C35) and

product (C40) in OPPs reaction. T. maritima OPPs enzyme (10 AM)

preincubated with FPP (2 AM) was mixed with [14C]IPP (50 AM) at pH 7.5

and 25 jC. The data represent the time courses of C20(D), C25(

.

), C30

(n), C35(y) and C40(E). The fitting curves were obtained from KINSIM

simulation using the kinetic pathway shown inScheme 1.

Plate 2. The intermediates and product formed during the T. maritima OPPs single-turnover reaction with 10 mM enzyme, 2 mM FPP and 50 mM [14C]IPP at

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ration with time during the single-turnover reaction(Fig. 3)

is obtained by using these kinetic constants to sum up all the intermediates in each specified time period of reaction. As shown in Scheme 1, the rate constant determined for each of the five IPP condensation steps leading to C40

product formation is approximately the same (f0.005 s 1) according to KINSIM simulation. Because this rate constant equals to the steady-state kcat value, the IPP condensation

represents the rate-limiting step of the T. maritima OPPs reaction. In contrast, the IPP condensation rate constant (2 s 1) of E. coli OPPs is 103times larger at 25 jC. However, at 80 jC, the T. maritima OPPs activity is greatly enhanced (Plate 2), consistent with the predicted increase of IPP condensation rate. This indicates that at high temperature, the IPP condensation is still a rate-limiting step for T. maritima enzyme.

3.3. Composition and secondary structure of T. maritima OPPs

In order to search for the possible cause of the significant reduction of OPPs activity at room temperature, we per-formed gel filtration chromatography to determine the composition of T. maritima OPPs and found that the enzyme is a dimer (data not shown). E. coli OPPs also is a dimeric enzyme and its dimerization is essential for product chain length determination [25]. Furthermore, the secondary structure of T. maritima OPPs measured using CD spectrophotometer is compared to that of E. coli enzyme. As shown in Fig. 5, both CD spectra are similar

with the a helix and h sheet contents of 52% and 23% for E. coli OPPs, and 59% and 26% for T. maritima OPPs, respectively, indicating the similar secondary structure for the two enzymes.

3.4. Product distribution of OPPs reaction

From the data presented above, under single-turnover condition C40is the exclusive product of T. maritima OPPs

reaction. We had further examined the enzyme final

prod-Chart 1. Kinetic constants used to simulate the time course of T. maritime OPPs single-turnover reaction.

Fig. 5. Analysis of secondary structures of T. maritima and E. coli OPPs enzymes using CD. The ellipticities of 1 AM E. coli enzyme (D) and 1 AM T. maritima enzyme (

.

) were recorded from 200 to 260 nm. Two enzymes have similar secondary structure according to their CD spectra.

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ucts after a long-time incubation (100 h) under a variety of different conditions. The results are shown inFig. 6and the quantitative data are summarized in Table 1. As shown in lane 1 of the figure, under multiple turnovers with 0.2 AM enzyme, 50 AM [14C]IPP and 50 AM FPP, the C20– C40

products were formed. This is due to that the IPP conden-sation rate is slow (f0.005 s 1) and FPP has a chance to displace the intermediates from the active site. Under the increased ratio of IPP to FPP (FPP concentration was reduced to 5 AM) as shown in lane 2, C40and very small

amount of C45were generated. The rate of C40conversion to

C45was extremely slow (210 5s 1). No further elongated

product larger than C45 could be observed even at high

temperature 80 jC where OPPs has enhanced activity (data not shown). An OPPs reaction with [EFPP]>[IPP] was performed to monitor the product formation with limited amount of IPP. The reaction shown in lane 3 of the figure contained 10 AM thermophilic OPPs, only 1 AM [14C]IPP and excessive FPP (50 AM). Under this condition where the EFPP concentration was 10-fold higher than that of IPP, C20-geanylgeranyl pyrophosphate was found as sole

prod-uct. The result remained the same when the reaction was performed at 80 jC (data not shown). This represents an

interesting difference between T. maritima OPPs and E. coli enzyme since the later generates C20– C40as products under

the same condition[14]. In a control reaction shown in lane 4, C40was synthesized by 10 AM T. maritima enzyme when

FPP concentration was reduced to 0.1 AM and [14C]IPP concentration remained as 1 AM.

4. Discussion

The OPPs encoding gene has been identified from the complete genome sequences of T. maritima using BLAST program [26]. This gave no firm answer regarding to the chain length of enzyme product as the sequence of T. maritima OPPs has 32%, 32% and 31% similarity with that of E. coli OPPs, Synechocystis sp. C45-solanesyl

pyrophop-shate synthase and Mycobacterium tuberculosis C35

-heptap-renyl pyrophosphate synthase, respectively (Fig. 1). We identify in this study the product of this putative enzyme to be C40. In order to know how a thermophilic enzyme is

different from the corresponding enzyme in a mesophile (E. coli), we conducted experiments to reveal the different properties of two enzymes at 25 jC. The kcatvalue of the

T. maritima OPPs reaction measured under steady-state condition is 0.005 s 1, consistent with the rate of IPP condensation obtained from enzyme single-turnover experi-ments. Therefore, the IPP condensation represents the rate-limiting step of the T. maritima OPPs reaction. In contrast, the IPP condensation catalyzed by E. coli OPPs is 2 s 1and the product release (steady-state rate) is 100-fold slower, indicating that the product release is rate determining. Despite the sequence homology between two enzymes, T. maritima OPPs has 103 times lower activity in IPP con-densation compared to E. coli OPPs at 25 jC. However, with increased temperature, the T. maritima enzyme has elevated level of activity. The activity of OPPs in T. maritima at high temperature is required for biosynthesis of menaquinone as a putative 1,4-dihydroxy-2-naphthoate octaprenyltransferase is identified in the genome of the bacterium. Indeed, most Gram-positive bacteria and anae-robic Gram-negative bacteria contain only menaquinone

[27]. The reason for causing 1000-fold lower activity in T. maritima OPPs at 25 jC must be due to a subtle change in the tertiary structure since the composition and secondary structure are similar for two enzymes.

Fig. 6. The product distribution of T. maritima OPPs reaction using FPP and [14C]IPP as substrates under various conditions for 100 h at 25 jC. The reaction conditions are lane 1: 0.2 AM enzyme with 50 AM [14C]IPP and 50 AM FPP; lane 2: 0.2 AM enzyme with 50 AM [14C]IPP and 5 AM FPP; lane 3: 10 AM enzyme with 1 AM [14C]IPP and 50 AM FPP; lane 4: 10 AM enzyme with 1 AM [14C]IPP and 0.1 AM FPP.

Table 1

Product distribution of T. maritima OPPs catalyzed condensation reactions of IPP with FPP under various conditions

Condition Product (%) C20 C25 C30 C35 C40 C45 0.2 AM E; 50 AM FPP; 50 AM IPP 28.0 22.3 7.6 11.9 28.3 1.8 0.2 AM E; 5 AM FPP; 50 AM IPP – – – 20.7 72.5 6.9 10 AM E; 50 AM FPP; 1 AM IPP 100 – – – – – 10 AM E; 0.1 AM FPP; 1 AM IPP – – – – 87.2 12.8

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The rate for an extra IPP incorporation into OPP is 0.02 s 1 and products with excessive IPP molecules were observed for E. coli OPPs[14]. However, for T. maritima OPPs, it is almost negligible (210 5s 1) for the formation of C45 and the larger polymers than C45 are not observed

even at high temperature where the T. maritima enzyme has higher activity. Apparently, T. maritima OPPs has higher product specificity compared to E. coli OPPs. Moreover, the product under the IPP single-turnover reaction of 10 AM T. maritima OPPsFPP complex with 1 AM IPP is C20

ger-anylgeranyl pyrophosphate at 25 jC and at high temperature (80 jC). In contrast, E. coli OPPs produces C20– C40

compounds under the same condition. The intermediate-bound OPPs from E. coli might undergo conformational change to increase its affinity with IPP leading to formation of product at lower concentration of IPP than OPPsFPP. The undecaprenyl pyrophosphate synthase that shows sim-ilar pattern of product distribution has a flexible structure as a protein conformational change was observed during cat-alysis[28]. However, this unique phenomenon was not seen in T. maritima OPPs.

In summary, we have utilized a pre-steady-state method to analyze the complete kinetic pathway of T. maritima OPPs reaction, which was not previously determined. This kinetic pathway contains five IPP condensation steps with a rate of f0.005 s 1 for each step simulated from our kinetic data. Despite the similar secondary structure, the enzyme has lower enzyme activity at 25 jC, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic E. coli counter-part.

Acknowledgements

This study was supported by a grant (NSC91-2113-M-001-007) from National Council of Science of Taiwan and a grant from Academia Sinica.

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數據

Fig. 1. Alignment of T. maritima OPPs (TC40) sequence with that of E. coli OPPs (C40), Synechocystis sp
Fig. 2. (A) T. maritima OPPs activity measured at different temperature.
Fig. 3. The single-turnover reaction of T. maritima OPPs with enzyme in excess of FPP
Fig. 5. Analysis of secondary structures of T. maritima and E. coli OPPs enzymes using CD
+2

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