Báo cáo khoa học: Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase doc

Effect of mutations at Glu160 and Val198 on the thermostabilityof lactate oxidase Hirotaka Minagawa1, Jiro Shimada1and Hiroki Kaneko2 1 Fundamental Research Laboratories, NEC Corp., Tsuk

Effect of mutations at Glu160 and Val198 on the thermostability

of lactate oxidase

Hirotaka Minagawa1, Jiro Shimada1and Hiroki Kaneko2

1

Fundamental Research Laboratories, NEC Corp., Tsukuba, Japan;2Department of Applied Physics, College of Humanities and Sciences, Nihon University, Tokyo, Japan

We have obtained two types of thermostable mutant lactate

oxidase – one that exhibited an E-to-G point mutation

at position 160 (E160G)through error-prone PCR-based

random mutagenesis, and another that exhibited an E-to-G

mutation at position 160 and a V-to-I mutation at position

198 (E160G/V198I)through DNA shuffling-based random

mutagenesis – both of which we have previously reported

Our molecular modeling of lactate oxidase suggests that the

substitution of G for E at position 160 reduces the

electro-static repulsion between the negative charges of E160 and

E130 in the (b/a)8barrel structure, but a

thermal-inactiva-tion experiment on the five kinds of single-mutant lactate

oxidase at position 160 (E160A, E160Q, E160H, E160R, and

E160K)showed that the side-chain volume of the amino acid

at position 160 mainly contributes to the thermostability of

lactate oxidase We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and ana-lysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes The half-life of E160G/V198I lactate oxidase at 70
C was about three times longer

2 than that of E160G lactate oxidase, and was about 20 times longer

3 than that of wild-type lactate oxidase In con-trast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation

Keywords: lactate oxidase; thermostability; site-directed mutagenesis; random mutagenesis

Lactate oxidase is widely used in biosensors to measure

the lactate concentration in blood [1], and increasing the

thermostability of lactate oxidase is a major factor in

prolonging the life of lactate oxidase-based lactate sensors

One way to achieve this is to use naturally thermostable

enzymes isolated from thermophilic bacteria; however, it is

not always possible to obtain a desired enzyme (e.g lactate

oxidase)from such bacteria An alternative method is to

enhance protein stability through protein engineering [2],

and many researchers have investigated the relationships

between protein structure and thermostability [3,4], with

some proposing a mutation strategy based on knowledge of

the specific enzyme, such as ribonuclease H [5] or lysozyme

[6,7], to improve enzyme stability by means of rational

design The thermostable thermolysin-like protease created

through rational design by van den Burg et al [8], was one

of the most successful cases, but we still need to obtain a

naturally thermostable counterpart thermolysin and

deter-mine its 3D structure The difficulty in trying to improve

enzyme stability through rational design is that this requires

solving the 3D structure of the enzyme, and, as the 3D

structure of the lactate oxidase has not yet been clarified, the

rational approach is therefore not applicable An alternative

approach to increase the enzyme thermostability is to screen mutants produced by random mutagenesis, such as error-prone PCR or DNA shuffling [9–11], for thermostable enzymes The technique of DNA shuffling was introduced

by Stemmer [9], and has become a powerful tool for protein engineering We have already produced two types of thermostable mutant lactate oxidase – E160G [12] and E160G/V198I [13] – and have used E160G lactate oxidase to develop a long-life lactate sensor [14] However, while we have shown that increasing the thermostability of lactate oxidase prolongs the life of lactate sensors, the thermo-stability of lactate oxidase must be further improved to enhance the practicality of its application to lactate oxidase sensors because the effect of enzyme stability tends to be weaker when the enzyme is immobilized on a membrane rather than in solution [14] We have constructed a 3D structure of lactate oxidase through homology modeling [15] In the case of E160G lactate oxidase, our model suggests that the Glu residue at position 160 in wild-type lactate oxidase is located in an a3 helix, constituting part

of the (b/a)8 barrel, and the mutation of Glu160 to Gly stabilized the entire protein structure by eliminating the negative charge repulsion between Glu160 and Glu130 To confirm this hypothesis, five kinds of single-mutant lactate oxidase (E160A, E160Q, E160R, E160K, and E160H)were constructed by site-directed mutagenesis, and their thermo-stability was compared with that of wild-type and E160G mutant lactate oxidases We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and compared the thermostability and Michaelis constant (K )values of wild-type, V198I, E160G, and E160G/V198I

Correspondence to H Minagawa, Fundamental Research

Laborat-ories, NEC Corp., Miyukigaoka 34, Tsukuba 305-8501, Japan.

Fax: + 81 29 856 6136, Tel.: + 81 29 850 2613,

E-mail: h-minagawa@ab.jp.nec.com

Abbreviation: IPTG, isopropyl thio-b- D -galactoside.

(Received 7 April 2003, revised 30 June 2003, accepted 14 July 2003)

lactate oxidases to investigate the effect of an E160G/V198I

double mutation

Materials and methods

Construction of the mutant lactate oxidase gene

The five types of single-mutant lactate oxidase at position

160 (E160A, E160Q, E160H, E160R, and E160K)were

constructed by PCR-based site-directed mutagenesis using

the Quick Change Site-Directed Mutagenesis Kit

(Strata-gene) According to the kit protocol, the plasmid containing

the mutant lactate oxidase gene was amplified by PCR with

125 ng of each mutagenic primer (forward and reverse)and

50 ng of pLODwt [12], as a template, under the following

conditions: after heat denaturation at 95
C for 30 s, we

applied 16 treatment cycles, each consisting of

30 s at 95
C, 1 min at 55
C, and 12 min at 68
C The

amplified plasmids were cooled on ice for 2 min and mixed

with DpnI at 37
C for 1 h Then 1 lL of plasmid was used

to transform Escherichia coli JM109 through

electropora-tion, and the electroporated JM109 cells were cultured

overnight at 37
C on an L-plate (1% tryptone, 0.5% yeast

extract, 0.5% NaCl, 1.5% agar, and 100 lgÆmL)1

ampicil-lin) Several transformant plasmids were sequenced and the

plasmid which had the intentional mutation was selected as

mutant lactate oxidase The E160G/V198I double-mutant

lactate oxidase was created by DNA shuffling [13], and the

V198I single-mutant lactate oxidase was created by

site-directed mutagenesis, as described above

Lactate oxidase purification and activity measurements

The wild-type and all mutant lactate oxidases were purified

by the method previously reported [12] E coli JM109 cells,

harboring a plasmid containing lactate oxidase genes, were

grown overnight in L-broth (1% tryptone, 0.5% yeast

extract, 0.5% NaCl, and 100 lgÆmL)1ampicillin)at 37
C

Isopropyl thio-b-D-galactoside (IPTG)was then added to a

final concentration of 1 mMand the cells were cultured for a

further 2 h The cells were then harvested and suspended in

a 50-mM potassium-phosphate buffer (pH 7.0)containing

1 mgÆmL)1 (w/v)lysozyme, and then stirred at room

temperature for 1 h After ultrasonic disruption of the cells,

a crude extract was obtained by centrifugation

Phenyl-methanesulfonyl fluoride and EDTA were added to the

supernatant as proteinase inhibitors, at final concentrations

of 1 mM

5 After precipitation by ammonium sulfate (60/80%

saturation)at room temperature, the lactate oxidase was

purified by stepwise column chromatography using

Q-Sepharose FF, Phenyl Sepharose 6FF, and Superdex

pg200 (all from Pharmacia)in the FPLC system

(Pharma-cia) The purity was verified by SDS/PAGE, using PhastGel

and the PhastSystem (Pharmacia) Lactate oxidase activity

was determined by a peroxidase-coupled

spectrophotomet-ric method [12] using H2O2as the standard Assays were

started by adding lactate oxidase to an activity assay

mixture containing 1.5 mM 4-aminoantipyrine, 3.3 mM

phenol, 100 mM L-lactate (lithium salt), 40 mMHepes, and

6 U of horseradish peroxidase, in a final volume of 2 mL at

pH 7.3, and then the absorbance (A)at 500 nm was

monitored at room temperature Protein concentrations

were measured with a bicinchoninic Protein Assay Regent Kit (Pierce)using purified bovine serum albumin as the standard Irreversible enzyme inactivation was measured at

a protein concentration of 50 lgÆmL)1in a 40 mMHepes buffer, pH 7.3 Samples were incubated at 70
C and then transferred to ice at different time-points during incubation Residual enzyme activity was then measured, as described above To determine the temperature dependence of the Km value forL-lactate at pH 7.3,L-lactate concentrations were varied over a significant range (78 lMto 10 mMor 0.78 mM

to 100 mM)and assays were conducted at 15, 25, 35, and

45
C Kmvalues were calculated by plotting [S]/V vs [S], where [S] and V are, respectively, theL-lactate concentration and the initial reaction rate

Results and discussion

Irreversible enzyme inactivation assay of mutant lactate oxidases at position 160

On the basis of our lactate oxidase model [15], position 160

is located in an a3helix constituting part of the (b/a)8barrel, which is a common structure and the most basic frame in the functionally important domain of a family of FMN-dependent a-hydroxy acid oxidizing enzymes The model suggests that even the shortest distance between Glu160 and Glu130 is 6.03 A˚ and this distance is probably a result of the electrostatic repulsion between the negative charges of these two glutamic acids We have explained that the partial disorder is caused by this negative charge repulsion between Glu160 and Glu130; the mutation of Glu160 to Gly (E160G: which was made by error-prone PCR [12])might therefore stabilize the entire protein structure by eliminating this charge repulsion [15] To test this hypothesis, we changed Glu160 to Gln (E160Q: the same side-chain volume and no electric charge), His, Arg, and Lys (E160H, E160R, E160K, respectively: positive electric charge) We also created mutant Ala at position 160 (E160A), which has a side-chain volume intermediate between those of Gln and Gly The thermal-inactivation curves of wild-type and mutant lactate oxidase at position 160 (E160G, E160A, E160Q, E160H, E160R, and E160K)at 70
C are shown in Fig 1: the heat-inactivation curve of the wild-type lactate oxidase exhibited a simple exponential decay, while those of mutant lactate oxidases exhibited biphasic inactivation This suggests that at least two processes were involved in the thermal inactivation of the mutants Similar phenomena have been previously observed for other thermostable mutants (N212D and E160G/N212D)[12] and may be explained by the multiple-exponential state in the unfolding kinetics [16] The relationships between biphasic inactivation

of the mutants and the enzyme thermostability are still not fully understood and therefore further study is required The thermostabilities of E160H, E160R, and E160K were almost the same as that of the wild-type lactate oxidase, and the thermostabilities of E160Q and E160A were slightly higher than that of the wild-type enzyme, but lower than that of the E160G mutant Substitution with a small, nonelectric amino acid at position 160 seems to affect the increase in lactate oxidase thermostability In the a-helix, Gly to Ala substitution was reported to stabilize the helix by decreasing the entropy of the unfolding state [17] In our

case, E160G was more thermostable than E160A, thus the

amino acid substitution at position 160 might have affected

the a3 helix flexibility through interaction with other

residues, such as Glu130 or Arg203 It is difficult to explain

the thermostability mechanisms of the E160G mutation on

the basis of this model structure, and the precise molecular

structure of lactate oxidase would have to be known to do

so Our group and other researchers [

determine the 3D structure of lactate oxidase, and

prelimi-nary results were obtained; however, the complete detailed

structure of lactate oxidase has not yet been determined

Irreversible enzyme-inactivation assay of mutant

lactate oxidases at positions 160 and 198

We created V198I single-mutant lactate oxidase by

site-directed mutagenesis using a Quick Change Site-Directed

Mutagenesis Kit (Stratagene)and purified the lactate

oxidase, as described above Thereafter, we compared the

irreversible thermostability and the temperature dependence

of the Kmvalue with the wild-type, E160G, and E160G/

V198I lactate oxidase The thermal-inactivation curve of the

E160G/V198I mutant at 70
C was obviously less steep

than that of the E160G mutant; however, there was very

little difference in the thermal-inactivation curves of the

V198I mutant and wild-type lactate oxidases (Fig 2) This

indicates that the V198I mutation affects the thermostability

of the E160G mutant lactate oxidase, but has a minimal

effect on the thermostability of the wild-type enzyme The

temperature dependence of the wild-type, E160G, V198I,

and E160G/V198I Km values for L-lactate are shown in

Fig 3 The temperature dependence of the K values of

Fig 1 Thermal inactivation of wild-type and mutant lactate oxidases at

position 160 Residual activities of wild-type (d), E160G (h), E160A

(n), E160Q (s), E160H (e), E160R (,), and E160K ( )lactate

oxidases are shown, expressed as a percentage of the original activity.

Enzymes in 40 m M Hepes buffer (pH 7.3)were incubated at 70
C for

different periods of time Results represent the mean value of

experi-ments performed in triplicate ± SD.

Fig 2 Thermal inactivation of wild-type, E160G, V198I, and E160G/ V198I lactate oxidases Residual activities of wild-type (d), E160G (h), V198I (s), and E160G/V198I (e)lactate oxidases are shown, expressed as a percentage of the original activity The enzymes, in 40-m M Hepes buffer (pH 7.3), were incubated at 70
C for different periods of time Results represent the mean value of experiments performed in triplicate ± SD.

Fig 3 Temperature dependence of the Michaelis constant (K m ) values

of wild-type, E160G, V198I and E160G/V198I lactate oxidases The K m

values of wild-type (d), E160G (h), V198I (s), and E160G/V198I (e) lactate oxidases, at 15, 25, 35, and 45
C, are shown The concentration

of L -lactate varied from 78 l M to 10 m M (wild-type and V198I)or 0.78 m M to 100 m M (E160G and E160G/V198I), and assays were conducted in 40 m M Hepes buffer at pH 7.3.

E160G and E160G/V198I were almost identical, and the

Kmvalues of these two mutants were higher than that of

the wild-type lactate oxidase at a temperature range of

15–45
C; thus, the Kmvalue and the temperature

depend-ence of the wild-type lactate oxidase were almost the same as

for the V198I mutant lactate oxidase In terms of the Km value, the E160G mutation greatly affected the lactate oxidase activity, but the V198I mutation had no or only a minor effect on the catalytic activity of lactate oxidase with respect to the wild-type and E160G lactate oxidases The

Fig 4 Stereoview of the model structure of lactate oxidase: a side view of the (b/a) 8 barrel The side-chains of Val198, Glu160, Arg203, Glu130, a pyruvate molecule and an FMN molecule are shown as a stick model The labels of the mutation sites, Glu160 and Val198 are colored green All the atoms of Gly126, Ala127 and Thr128 are displayed by a van der Waals surface (CPK)model The atom-type colors are as follows: carbon (green), oxygen (red), nitrogen (blue) and phosphate (magenta) The region considered a flexible large loop structure (residues 189–215), on the basis of the structural comparison between glycolate oxidase and flavocytochrome b 2 , is shown as a blue ribbon (see also the thick lines in Fig 5) Red cylinders and yellow arrows represent a-helixes and b-strands, respectively, with the a 2 , a 3 , a D and a E helices denoted by a2, a3, aD and aE, respectively The secondary-structure nomenclature is according to Lindqvist [19] The picture was created using INSIGHT II software (Version 4.3, Accelrys Inc.)on an ONYX2 workstation (Silicon Graphics, Inc.).

Fig 5 Variation of the atomic temperature factors averaged for the main-chain atoms of spinach glycolate oxidase [19] The data were obtained from the Protein Data Bank (entry ID: 1GOX) The average B-factor of each residue is plotted against the residue number Residues 172–204, in which even the main-chain structures are not superimposed between glycolate oxidase and the FMN-binding domain of flavocytochrome b 2 , are emphasized by thick lines Residues 189–197, which are disordered in the crystal, are shown as dotted lines Only the a D helix, a E helix and the secondary structures included in the (b/a) 8 barrel are indicated The left arrow indicates the value for Arg143 (corresponding to Glu160 of lactate oxidase), and the right arrow indicates the value for Lys181 (corresponding to Val198 of lactate oxidase).

specific activity of V198I lactate oxidase was approximately

the same as that of wild-type lactate oxidase, and that of

E160G/V198I mutant lactate oxidase was approximately

the same as that of E160G (data not shown) These

observations suggest that the V198I mutation only affects

the thermostability of the enzyme when it combines with

the E160G mutation

Why does the single E160G mutation affect the

thermo-stability whereas the V198I mutation does not? According

to our lactate oxidase model [15], position 160 is located in

an a3helix constituting part of the (b/a)8 barrel (Fig 4),

which is a common structure and the most basic frame in

the functionally important domain of a family of

FMN-dependent a-hydroxy acid oxidizing enzymes The atomic

temperature factors of the corresponding position

(B-value¼ 25 A˚2)in glycolate oxidase suggest that

posi-tion 160 is not flexible, whereas the average isotropic

B-value for all the main-chain protein atoms is 26.5 A˚2

(Fig 5)[19] Position 198, on the other hand, is located in a

large loop (residues 189–215)

loop as the region corresponding to the place where the

main-chain structures of two homologous enzymes –

glycolate oxidase [19] and the FMN-binding domain of

flavocytochrome b2[20] – are not superimposed on the basis

of the 3D structural fitting The loop starts just after the end

of the aDhelix and terminates halfway along the aEhelix,

thus it is quite far from the core structure [i.e the (b/a)8

barrel] Moreover, the atomic temperature factors of the

corresponding position in glycolate oxidase (Fig 5)suggest

that the loop which includes position 198 is very flexible

We infer from this that position 160 contributes more

directly to stabilizing the (b/a)8barrel structure than does

position 198 Therefore, the mutation at position 160

should have a much greater effect on stability than the

mutation at position 198

Why does the V198I mutation further increase the

thermostability of the E160G mutant? Glu160 in the

wild-type lactate oxidase corresponds to Arg143 in the a3helix of

glycolate oxidase From detailed observation of the

glyco-late oxidase crystal structure [19], we have learned that

Arg143 plays a very important role in maintaining the

stability of the enzyme molecule [15] The residue stabilizes

the entire (b/a)8 barrel structure by forming a strong salt

bridge with the Glu114 in the a2helix It also reinforces the

interaction between the (b/a)8barrel structure and the large

loop, mentioned above (residues 172–204 in glycolate

oxidase and residues 189–215 in the wild-type lactate

oxidase), by forming a hydrogen-bond network between

the NH2of Arg143 and the main-chain carbonyls of Lys181

and Asn182 directly, and the main-chain carbonyl of

Glu184 by way of a water molecule However, in lactate

oxidase, the exchange of the E160G mutation appears to

completely eliminate the interaction between the (b/a)8

barrel structure and the large loop that would otherwise

occur through the hydrogen bond network between Glu160

in the barrel and Arg203 in the loop, in return for stabilizing

the barrel structure (see Fig 4) A Valfi Ile mutation at

position 198 in the large loop may restore the interaction

Actually, according to our lactate oxidase model (Fig 4),

the extension of the side-chain generated by the Valfi Ile

mutation forms van der Waals contacts with the side-chain

atoms of Thr128 or the main-chain atoms of Gly126 and

Ala127 As residues 126–128 are located just before the a2 helix, it is probable that the Valfi Ile mutation at position

198 reinforces the interaction between the (b/a)8 barrel structure and the large loop via van der Waals forces between the Ile side-chain and the side-chains of amino acids near the a2helix

The additive effects of mutations on enzyme stability have often been reported [21] Hence, one strategy to improve enzyme stability is to screen several thermostabi-lized mutant enzymes that have a single amino acid mutation, and then to generate a combined mutant that has two or more amino acid mutations We have previously applied this additive strategy to improve the thermostabi-lity of lactate oxidase [12] In that work we obtained two types of thermostable mutant lactate oxidase (N212D and E160G)through random mutagenesis and constructed a double mutant N212D/E160G lactate oxidase The thermo-stability of that double mutant, however, was approxi-mately the same as that of E160G lactate oxidase [12] In this case, we found that the N212D mutation affects the enzyme stability of wild-type lactate oxidase, but not that of E160G lactate oxidase Although many researchers have found that combinations of mutations, and the resulting effects of the amino acid substitutions at different positions, are usually additive, our findings for two types of double mutations (N212D/E160G and E160G/V198I)indicate that the cooperative amino acid substitutions also have an important effect on enzyme thermostability

Acknowledgements

We thank M Kitabayashi of NEC’s Fundamental Research Labor-atories for her technical assistance in the sample preparation. References

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