Engineering a chimeric pyrroloquinoline quinone glucose ...

Protein Engineering vol.12 no.1 pp.63–70, 1999

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

Hiromi Yoshida, Katsuhiro Kojima, Arief Budi Witarto and Koji Sode1 Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan 1To

whom correspondence should be addressed

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A.calcoaceticus PQQGDH(A27 region) and also corressponds to the same position from 32 to 59% from the N-terminus in E.coli PQQGDH, though E.coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A.calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E.coli PQQGDH resulted in an increase in the substrate specificity of E.coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, β-propeller structure. Keywords: PQQ glucose dehydrogenase/chimeric enzymes/ biosensor/substrate specificity/thermal stability Introduction A variety of enzymes possessing pyrroloquinoline quinone (PQQ) as their prosthetic group has been found in Gram © Oxford University Press

negative bacteria (Anthony, 1992), such as ethanol dehydrogenases (EDHs), methanol dehydrogenases (MDH) and glucose dehydrogenases (GDHs). Recently, the three-dimensional (3D) structure of PQQMDH was elucidated (White et al., 1993; Ghosh et al., 1995). PQQMDH is a β-propeller protein composed of eight W-motifs. Each W-motif is composed of four antiparallel β-strands. Considering the typical W-motif patterns in the primary structure within PQQ enzymes, PQQEDHs and PQQGDHs have also been predicted to be βpropeller proteins (Cozier et al., 1995; Cozier and Anthony, 1995). It is expected that PQQ enzymes will be utilized for the construction of a biochemical monitoring system for organic compounds. In particular, extensive attention has been paid to PQQGDHs as an enzyme sensor constituent for glucose monitoring (Turner et al., 1987; Yokoyama et al., 1989; Sode et al., 1993; Ye et al., 1993). In addition, the application of PQQGDH in diagnostics is highly valued because it can be carried out independent of oxygen (Schmidt, 1997). Two types of PQQGDH have been found in a variety of Gram-negative bacteria. One is a soluble PQQGDH (PQQGDH-B) which has only been found in A.calcoaceticus. PQQGDH-B is a homodimeric enzyme consisting of two identical subunits of approximately 50 kDa and its sequence has no significant homology with any other proteins so far reported (Dokter et al., 1986; Cleton-Jansen et al., 1988; Anthony, 1992). The biological role for PQQGDH-B is still unknown. Membranebinding PQQGDHs (PQQGDH-A) are widely distributed among many bacterial species and they are all single peptides with molecular weights of about 87 kDa. In spite of their highly homologous primary structures, the enzymatic characteristics, such as co-factor binding stability, thermal stability and substrate specificity, are dependent upon the derived bacterial sources. Although its fine tertiary structure has not yet been elucidated, the restriction of the protein region responsible for each enzymatic characteristic will be carried out by extensive homology analyses together with mutational analyses. The authors have used a protein engineering approach in order to elucidate these regions and ultimately to construct an ideal PQQGDH for diagnostic usage, which possesses high co-factor binding stability, high thermal stability and adequate substrate specificity. We have been focusing on one set of PQQGDH structural genes, Escherichia coli PQQGDH, which loses its prosthetic group, PQQ, in the presence of EDTA, and A.calcoaceticus PQQGDH (PQQGDH-A), which is very stable during EDTA treatment (Sode et al., 1995). The reversible inactivation procedure by the EDTA treatment has been recognized as a result of the chelating of the divalent metal ion which is essential for the holo-enzyme formation. Therefore, the EDTA tolerance can be interpreted as an indicator of the co-factor binding stability. Based on the construction of a variety of chimeric enzymes between E.coli and A.calcoaceticus PQQGDHs, we have elucidated the main region responsible for EDTA tolerance in A.calcoaceticus PQQGDH (Sode et al., 1995b). The region was composed of about 90 amino 63

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Fig. 1. Construction of chimeric PQQGDH, E32A27E41 by homologous recombination.

acid residues and located between 45 and 56% of the distance from the N-terminal region in A.calcoaceticus PQQGDH. According to the information, we constructed a chimeric PQQGDH, E45A14E41, by substituting the 14% region (between 45 and 59% from the N-terminal region) in E.coli PQQGDH with the corresponding 14% region from A.calcoaceticus PQQGDH. E45A14E41 showed a significant increase in the EDTA tolerance as compared with E.coli PQQGDH. However, it was not the same level as A.calcoaceticus PQQGDH (Sode and Yoshida, 1997). This result indicated that our reported region was not enough to show the complete increase in EDTA tolerance. Probably some other regions exist which may coordinately contribute to the EDTA tolerance together with the elucidated region. One of the constructed chimeric enzymes, designated as E97A3, of which the N-terminal 97% region is from E.coli and the remaining 3% from A.calcoaceticus PQQGDH, showed an increase in thermal stability (Sode et al., 1995a). The Cterminal 3% region from A.calcoaceticus PQQGDH played an important role in the increase in the thermal stability with the combination of the N-terminal region of E.coli PQQGDH. We recently reported the improvement of the substrate specificity of E.coli PQQGDH by substituting His775 to Asn (Sode and Kojima, 1997). The amino acid residue may strongly affect the substrate specificity by interacting with the C6 hydroxy group of saccharides. These findings encourage us to construct an enzyme composed of all the regions responsible for the improvement of enzymatic characteristics, which may lead to the construction of an ideally engineered enzyme. In addition, research into the compatibility of these protein regions is also essential. In this study, we first investigated the complete region encoding EDTA tolerance in A.calcoaceticus PQQGDH. Based on this finding, we constructed a variety of chimeric and mutant PQQGDHs composed of a combination of the following three different protein regions responsible for EDTA tolerance, improvement of substrate specificity and increase in the thermal stability. 64

Fig. 2. Genetic construction of PQQGDHs used in this study. m, derived from A.calcoaceticus PQQGDH structural gene; u, derived from E.coli PQQGDH structural gene. a), b), restriction enzyme sites on the expression vector, pTrc99A. H775N, H782N, the amino acid substitution at either His775 or His782 to Asn.

Materials and methods Bacterial strains and plasmids The bacterial strains and plasmids used in this study are listed in Table I. Escherichia coli JC8679 was used for the construction of chimeric PQQGDH by homologous recombination. Escherichia coli PP2418, where the PQQGDH structural gene has been disrupted by insertion mutagenesis (Cleton-Jansen et al., 1990), was used as the host strain for the expression of each PQQGDH. Escherichia coli BMH7118mutS and E.coli MV1184 were used for constructing mutants by site-directed mutagenesis. All the PQQGDH structural genes, including chimeric PQQGDHs and mutated PQQGDHs, were inserted into the multi-cloning site of the expression vector, pTrc99A (Pharmacia, Sweden). Construction of chimeric and mutated PQQGDHs Figures 1 and 2 show the construction of the chimeric and mutated PQQGDHs in this study. The junction between E.coli PQQGDH and A.calcoaceticus PQQGDH, and the mutated position in chimeric PQQGDHs are indicated in the sequence alignment of E.coli PQQGDH and A.calcoaceticus PQQGDH (Figure 3). The position of amino acid residues are defined relative to the initiator methionine residue of the constructed chimeric PQQGDHs. His775 in E.coli PQQGDH corresponds to His781 in A.calcoaceticus PQQGDH. Therefore, according to this definition, His775 of E.coli PQQGDH corresponds to His782 in chimeric PQQGDHs harbouring the A27 region. E32A27E41 E32A27E41 was constructed on the basis of the homologous recombination as previously described (Sode et al., 1995b) (Figure 1). The structural genes of chimeric PQQGDHs, E32A68 (32% of the E.coli PQQGDH N-terminal region and 68% of the A.calcoaceticus PQQGDH C-terminal region) and E10A49E41 (10% of the N-terminal and 41% of the C-terminal region of E.coli PQQGDH and the remaining 49% from

Engineering a chimeric PQQGDH

Table I. List of strains and plasmids Strains or plasmids Strain JC8679 PP2418 BMH71–18 mutS MV1184 Plasmid pTrc99A pGEcI pGEcI H775N pGE97A3 pGE32A68 pGE10A49E41

Genotype or phenotype

Reference or source

recBC, sbcA ptsI, thi, galP, gcd::cat ∆(lac-proAB), thi, supE, mutS215::Tn10(tet r )/F9, traD36, proAB, lacIq, lacZ∆M15 ∆(lac-proAB), ara, strA, thi, (ø80 lacZ∆M15), ∆(srl-recA)306::Tn10 (tetr)/F9, traD36, proAB, lacIq, lacZ∆M15

Gillen et al., 1981 Cleton-Jansen et al., 1990 Takara Takara

Apr Apr, Apr, Apr, Apr, Apr,

Pharmacia Sode and Sano, 1994 Sode and Kojima, 1997 Sode et al., 1995 Sode and Sano, 1995b Sode et al., 1995b

pTrc99A pTrc99A pTrc99A pTrc99A pTrc99A

derivative derivative derivative derivative derivative

containing containing containing containing containing

E. coli PQQGDH structural gene mutant pGECI chimeric PQQGDH E97A3 structural gene chimeric PQQGDH E32A68 structural gene chimeric PQQGDH E10A49E41 structural gene

Fig. 3. Alignment of sequences of E.coli PQQGDH and A.calcoaceticus PQQGDH. u, shows A27 region responsible for EDTA tolerance; dotted line, A3 region responsible for thermal stability. His775 of E.coli PQQGDH and His781 of A.calcoaceticus PQQGDH are the sites responsible for substrate specificity.

A.calcoaceticus PQQGDH) were used (Sode et al., 1995b). The plasmid containing E32A68 was digested with PstI and blunt-ended. The NcoI fragment of plasmid pGE10A49E41, containing the chimeric PQQGDH region, was ligated with the blunt-ended plasmid, resulting in a plasmid containing two chimeric PQQGDH structural genes inserted in tandem. After a MscI/HindIII double digestion, linearized plasmid was transformed into E.coli JC8679 (recBC, sbcA) for the homologous recombination. Plasmids from the transformants were then extracted, and transformed into PP2418. Transformants showing PQQGDH activity were picked up and their plasmids containing chimeric enzyme were analysed. The nucleotide sequence of the chimeric region was confirmed to be

E32A27E41 using an automated DNA sequencer (Shimadzu DSQ-1, Japan). E97A3 H775N E97A3 H775N was constructed on the basis of the structural gene of a chimeric PQQGDH, E97A3, by substituting His775 to Asn (Figure 2). pGE97A3 was digested with AvaI and PstI, and the fragment inserted into linearized pKF18K (Takara, Japan) digested with AvaI and PstI. Using the synthesized oligonucleotide primer 59-CCAAATGAACCATTACCACCGGCCA-39 site-directed mutagenesis was performed according to the instruction manual of the Mutan-Super Express Km kit (Takara, Kyoto, Japan). The nucleotide sequence of the mutation was confirmed by an automated DNA sequencer. Thus, 65

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the resultant mutated region was digested with AvaI and PstI, and the fragment was substituted with the corresponding region of pGE97A3, thus forming E97A3 H775N. E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N Based on the structural genes of E32A27E41, new chimeric PQQGDHs, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed (Figure 2). The structural gene of E32A27E41 inserted in pTrc99A was digested with PvuI (one site is located within the gene and the other on the vector), and was ligated with the PvuI fragments of pGEcI H775N, pGE97A3 or E97A3 H775N structural genes inserted into pTrc99A. This procedure resulted in the substitution of the E32A27E41 C-terminal region with each of the C-terminal regions of other chimeric and mutated PQQGDH structural genes, to construct E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N, respectively. Enzyme purification The expression vector containing each chimeric or mutated PQQGDH structural gene was transformed into E.coli PP2418, and cultivated according to the previous study (Sode and Sano, 1994). The membrane fraction obtained according to the previous study was solubilized in the presence of 1% Triton X-100 in a 10 mM potassium phosphate buffer, pH 7.0 for 30 min, at a temperature of 4°C. The prepared solubilized membrane fraction was then subjected to ultracentrifugation (160 500 g, 1.5 h, 4°C), followed by dialysis in 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. The supernatant was used as the crude enzyme preparation. The crude enzyme preparation was applied to a DEAE 2025 column (Showa denko, Japan) equilibrated with 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. After the column was washed with the same buffer (2 column volumes), the enzyme was eluted with a linear gradient of 0–0.1 M KCl in 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. The fractions showing enzyme activity were applied to the DEAE 5PW column (TOSOH, Japan) and eluted with a linear gradient of 0–0.225 M NaCl in a 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. The purified enzyme found to be electrophoretically homogeneous by silver staining on SDS–PAGE was used for the kinetic studies. Analysis of EDTA tolerance of PQQGDHs EDTA tolerance of each enzyme was analysed by using crude enzyme preparation as samples. The enzyme was dialyzed against 10 mM MOPS (3-[N-morpholino] propanesulfonic acid)–KOH buffer, pH 7.0, containing 0.2% Triton X-100 and incubated in the same buffer in the presence of 5 µM PQQ, 1 mM MgCl2 or 1 mM CaCl2 at 25°C to form the holoenzyme. After that, samples were incubated in the presence of 10 mM EDTA, and the time course of the residual enzyme activity was measured. PQQGDH activity was assayed as described previously (Sode et al., 1995b). Analysis of thermal stability of PQQGDHs Thermal inactivation was measured by incubating 56 µl of the enzyme solution in 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100 at each temperature for 10 min. Samples were then stored at 4°C for 1 min, and the residual enzyme activity was determined after the holo-enzyme had formed in 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100, 10 mM MgCl2 and 5 µM PQQ for 1 h. The residual activity was compared with the initial 66

activity. The half-lives of thermal inactivation (t1/2) of the enzymes were determined by measuring the time course of thermal inactivation at 45°C. Samples were taken every 2 or 5 min, and all other experimental conditions were the same as above. t1/2 was obtained by linear regression in semilogarithmic coordinates. Analysis of substrate specificity of PQQGDHs The relative activities towards each substrate were obtained by comparison with the activity for glucose as a control at 1 mM using crude enzyme preparations as enzyme samples. Kinetic parameters were determined by using purified enzymes. Enzyme samples were incubated in a 10 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100, 10 mM MgCl2 and 5 µM PQQ to form the holo-enzyme. In the presence of 0.6 mM phenazine methosulfate, 0.06 mM 2,6-dichrolophenolindophenol (DCIP) at 25°C, various concentrations of the substrate were injected, and the decrease in absorbance of DCIP was measured at 660 nm. Circular dichroism (CD) Far-UV CD spectra of E.coli wild-type PQQGDH and chimeric PQQGDHs were measured with a spectropolarimeter, model J720 (JASCO, Tokyo, Japan) at room temperature. A cylindrical quartz cell with a path-length of 0.02 cm (JASCO, Tokyo, Japan) was used. For CD measurements, chimeric PQQGDH samples with a concentration of 600 µg ml–1 were used. CD spectra were measured by scanning the samples three times from 260 to 184 nm with an increment of 0.2 nm, at a speed of 20 nm min–1. The CD spectra were averaged and subtracted with the appropriate buffer spectra. Secondary structure contents were estimated using Convex Constraint Analysis (CCA) (Perczel et al., 1991) with a collection of 30 membrane protein CD spectra as references (Park et al., 1992). Results EDTA tolerance of mutant E.coli PQQGDHs According to our previous study (Sode and Yoshida, 1997), E45A14E41, containing the region responsible for EDTA tolerance, did not reach the same level of EDTA tolerance as A.calcoaceticus PQQGDH, though it was certain to increase the EDTA tolerance more than E.coli PQQGDH. Considering that E32A68 showed complete EDTA tolerance but E56A44 did not (Sode and Yoshida, 1995b), we constructed a new chimeric PQQGDH in order to extend the region between 45 and 59% of E45A14E41 to between 32 and 59%. Constructed chimeric PQQGDH, E32A27E41 is composed of 27% of the A.calcoaceticus PQQGDH region (A27 region) located from the 32% from the N-terminal region. Figure 4 shows the time courses of the inactivation procedure of PQQGDHs in the presence of 10 mM EDTA. Although E.coli PQQGDH was readily inactivated in the presence of EDTA within 20 min, the constructed chimeric enzyme, E32A27E41 showed EDTA tolerance in the same way as A.calcoaceticus did. The observed EDTA tolerance was much higher than E45A14E41 chimeric GDH (30% residual activity after 30 min incubation in the presence of 10 mM EDTA) as previously reported by Sode and Yoshida (1997b). This result suggested that the A27 region contains a complete region responsible for EDTA tolerance. We also investigated the EDTA tolerance of other E.coli PQQGDH mutants. All the mutants harbouring the A27 region showed EDTA tolerance as was observed in A.calcoaceticus

Engineering a chimeric PQQGDH

Fig. 4. EDTA tolerance of PQQGDHs. In the presence of 10 mM EDTA at 25°C, time courses of residual activity were measured. The initial activities showed in the absence of EDTA were used as a control. s, E.coli PQQGDH; u, A.calcoaceticus PQQGDH; d, E32A27E41; j, E32A27E38A3 H782N.

PQQGDH (data not shown). E32A27E38A3 H782N, which was constructed from all the regions responsible for the target enzymatic characteristics, also showed EDTA tolerance (Figure 4). Therefore, the function encoded in the A27 region was found to be compatible with all the other regions. Thermal stability Figure 5a shows the thermal stability of PQQGDH mutants harbouring the His775 to Asn (H775N) substitution. H775N showed a marked decrease in thermal stability compared with its parental enzyme, EcI. The H775N substitution improved the substrate specificity but simultaneously caused the decrease in thermal stability. We previously reported that chimeric PQQGDH, E97A3, showed a significant increase in thermal stability as compared with EcI. Therefore, we investigated the compatibility of these two regions, the N-terminal 3% region from A.calcoaceticus PQQGDH (A3 region) and the His775 to Asn substitution (Figure 5a). The constructed mutant enzyme, E97A3 H775N, showed much higher thermal stability than H775N, and also higher than EcI. Consequently, the A3 region was able to complement the decrease in the thermal stability caused by the substitution of His775 with Asn. Figure 5b shows the thermal stability of chimeric enzymes harbouring the A27 region. As was observed in the wild-type PQQGDH EcI and H775N, the substitution of the amino acid residue His to Asn corresponding to His782 in E32A27E41, resulted in a decrease in thermal stability. The half-lives of thermal inactivation at 45°C (t1/2) for E32A27E41 and E32A27E41H782N were 27 and 7.7 min, respectively. However, the introduction of the A3 region into E32A27E41 H782N resulted in an increase in thermal stability (t1/2 5 21 min). As a result, E32A27E38A3 H782N also showed high thermal stability as E32A27E41 or E32A27E38A3 (t1/2 5 39 min). It is also remarkable that the chimeric enzymes harbouring the A27 region showed higher thermal stability than those without. The enzymes without the A27 region showed t1/2 less than 2 min, whereas E32A27E41 showed t1/2 higher than 39 min. Circular dichroism spectroscopy Far-UV CD spectra of E.coli wild-type PQQGDH (Witarto et al., 1998), E97A3 chimeric enzyme and E32A27E38A3

Fig. 5. Thermal stability of PQQGDHs. (a) d, E97A3 H775N; s, EcI; j, H775N. (b) s, E32A27E41; u, E32A27E41 H782N; d, E32A27E38A3; j, E32A27E38A3 H782N. After 10 min of incubation at each temperature, the residual activities were determined.

H782N chimeric enzyme are shown in Figure 6. The amount of secondary structure of these enzymes as determined by spectral analysis is also shown in Table II. Far-UV spectra of E97A3 chimeric enzyme is virtually identical with E.coli wildtype PQQGDH, and the secondary structure content is also identical. However, the CD spectrum of the E32A27E38A3 H782N chimeric enzyme is distinct from that of E97A3, and consequently the secondary structure content is different from those of wild-type and E97A3 chimeric enzymes. Instead of the decrease in the β-sheet content of E32A27E38A3 H782N, the random structure content increased compared with that of E97A3 and wild-type enzymes. Substrate specificity Table III summarizes the substrate specificity of various PQQGDHs. In addition to glucose, E.coli PQQGDH is able to 67

H.Yoshida et al.

oxidize 2-deoxy-D-glucose (2-dG). However, the substitution of His775 to Asn resulted in an increase in the substrate specificity, and a decrease in the relative activity toward 2-dG. PQQGDH, which has no substitution of His to Asn, showed similar substrate specificity as EcI, and oxidized 2-dG effectively. In contrast, all those mutants whose His residues were substituted to Asn, increased substrate specificity as did H775N, indicating that relative activities for 2-dG were less than 40% as compared with glucose. Therefore, the increase in substrate specificity by substitution of His to Asn was compatible with the region responsible for EDTA tolerance (A27 region) and for the increase in thermal stability (A3 region). Table IV summarizes the kinetic parameters of the major mutant PQQGDHs used in this study. Although E32A27E41 gained EDTA tolerance, the introduction of the A27 region decreased the catalytic activity judging by the Vmax/Km value. E32A27E38A3 H782N showed a similar Vmax/Km value for glucose as E32A27E41, but showed higher Km and Vmax values

Fig. 6. Far-UV CD spectra of E.coli PQQGDH, E97A3 and E32A27E38A3H782N. Solid line, E.coli PQQGDH and E97A3; dotted line, E32A27E38A3 H782N. PQQGDH samples with a concentration of 600 µg ml–1 were used.

Table II. Secondary structure contents (%) of chimeric PQQGDHs

E. coli PQQGDH E97A3 E32A27E38A3 H782N

Trans-membrane Helix helix

Sheet

Turn

Random

17.5 17.5 17.3

35.2 35.2 32.8

7.0 7.0 7.2

36.3 36.3 39.1

4.0 4.0 3.6

as compared with E32A27E41. Such an alteration in both Km and Vmax values was also observed when the substitution of His775 to Asn was introduced into EcI. When the kinetic parameters are compared between glucose and 2-dG, the Km values of H775N and E32A27E38A3 H782N in 2-dG were about 10-fold higher than those in glucose, though their Vmax values in 2-dG were less than in glucose. Consequently, these mutants showed high substrate specificity for glucose judging by the Vmax/Km values. These results show that the constructed E32A27E38A3 H782N mutant had EDTA tolerance and increased in both thermal stability and substrate specificity as compared with the parental enzyme, and it was simultaneously concluded that the regions responsible for enzymatic characteristics were compatible. Discussion In this paper, we have reported on the compatibility of the protein region responsible for the improvement of the enzymatic properties of PQQGDH, and the construction of an enzyme which was composed of all of those regions. We first investigated and restricted the fine region responsible for EDTA tolerance of PQQGDH, and as a result, succeeded in the construction of a chimeric PQQGDH, E32A27E41, with complete EDTA tolerance as found in A.calcoaceticus. Considering that E32A68 showed EDTA tolerance, but E10A35E55 did not (Sode and Yoshida, 1995b), the region located between 32 and 45% of the distance from the N-terminal region in A.calcoaceticus alone did not contribute to the EDTA tolerance. Considering that E45A14E41 has partially gained EDTA tolerance (Sode and Yoshida, 1997) and that E32A2TE41 was completely EDTA tolerant, the function encoded in the region between 32 and 45% may coordinately express EDTA tolerance with the region between 45 and 59%. All chimeric enzymes harbouring the A27 region showed the same EDTA tolerance as shown by A.calcoaceticus PQQGDH. Since the introduction of the A27 region did not have a negative effect on both the thermal stability and substrate specificity, the A27 region was found to be compatible with other regions responsible for the improvement of enzymatic properties. The introduction of the A27 region resulted in an unexpected increase in the thermal stability, and it was more effective than the introduction of the A3 region. However, it is also obvious that the A27 region could not complement the decrease in thermal stability caused by the His782 to Asn substitution, considering that t1/2 is for E32A27E41 (27 min), E32A27E41 H782N (7.7 min) and for E32A27E38A3 H782N

Table III. Enzyme activity of PQQGDHs Relative activity

D-Glucose 2-deoxy-D-glucose D-Mannose D-Allose D-Galactose D-Xylose Maltose

EcI

H775N

E97A3

E97A3 H775N

E32A27E41

E32A27E38A3

E32A27E41 H728N

E32A27E38A3 H782N

100% 69% 5% 81% 8% 21% 3%

100% 11% 0% 4% 0% 2% 2%

100% 76% 4% 93% 7% 10% 0%

100% 27% 0% 29% 0% 0% 0%

100% 69% 7% 92% 18% 37% 4%

100% 61% 11% 87% 17% 35% 6%

100% 31% 13% 58% 15% 17% 13%

100% 18% 1% 46% 7% 1% 7%

Each enzyme activity was measured at 1 mM substrate concentration.

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Engineering a chimeric PQQGDH

Table IV. Kinetic parameters of PQQGDHs Glucose

EcI H775N E32A27E41 E32A27E38A3 H782N

2-deoxy-D-glucose

Km (mM)

Vmax

Vmax/km

Km (mM)

Vmax

Vmax/km

0.9 1.5 0.9 4.4

110 417 6.6 26.5

122 (100%) 278 (100%) 7.3 (100%) 6.0 (100%)

1.2 13 – 30.2

87.6 256 – 24.0

73.0 (60%) 19.7 (77%) – 0.8 (13%)

Vmax is expressed in U/mg protein. –: not tested.

Fig. 7. Predicted secondary structure topology of chimeric PQQGDH, E32A27E38A3 H782N. Solid arrows and lines are derived from A.calcoaceticus PQQGDH. Open arrows and gray lines are derived from E.coli PQQGDH. A27 region corresponding to the region responsible for EDTA tolerance. A3 region corresponding to the region responsible for thermal stability. Each arrow shows β-strand composing of W motif. W1~W8, each W motif; N, N-terminus; C, C-terminus; H782N, His782 was substituted with Asn, the amino acid residue responsible for the substrate specificity. D354, N355, T424, putative divalent metal ion binding sites; D466, K493, putative active sites.

(21 min). Therefore, the role of the A27 region in thermal stability is different from that of the A3 region. CD spectroscopic analysis revealed that the chimeric enzyme harbouring the A27 region showed a different secondary structure content from that of the wild-type and E97A3 chimeric enzymes. The increase in the random structure is consistent with that of chimeric PQQGDH harbouring the A27 region which showed a decrease in enzyme activity. The increase in thermal stability may be the result of the rigid structure of the enzymes harbouring the A27 region. Figure 7 shows the secondary structure topology of the constructed chimeric PQQGDH, E32A27E38A3 H782N, which is composed of eight W-motifs, according to the predicted 3D structure. From the predicted motif of PQQGDH, the A27 region is located in the middle of the enzyme, and includes the W2, W3 and W4 motifs derived from A.calcoaceticus PQQGDH. Each W motif is composed of four consecutive anti-parallel β-strands (a-strand to d-strand). These three W motifs include the putative divalent metal ion binding sites (Asp354, Asn355 and Thr424) (Cozier and Anthony, 1995). The A27 region also includes a part of the consecutive inter motif region including Asp466, which is

proposed to be an active site base initiating reaction with the substrate by proton abstraction (Cozier and Anthony, 1995). Another putative active centre, Lys493, which forms salt bridges with Asp466 and both PQQ O5 and O4, is located in the W5 motif, between the W5b-strand and W5c-strand. In the case of the chimeric enzyme harbouring the A27 region, the W5 motif is derived from E.coli PQQGDH. Supposing that W4 and W5 motifs coordinately form the active site cavity composed of both Asp466 and Lys493, both the flexible movement of these regions and the interaction between W4 and W5 motifs might play a significant role in the catalytic activity of PQQGDH. Comparing the homology of the 30 amino acid residues corresponding to W4d-strand, W5a-strand and their inter motif region of A.calcoaceticus PQQGDH with those of E.coli PQQGDH, nine amino acids are substituted and one insertion is observed. It would be possible, therefore, that such amino acid substitutions may be responsible for the alteration of the interaction between W4 and W5 in the chimeric enzymes harbouring the A27 region, consequently resulting in a decrease in catalytic activity. Therefore by optimizing the interaction between W4 and W5 motifs, the construction of an EDTA tolerant PQQGDH without decrease in the catalytic activity will be achieved. We recently reported that His775 substitution to Asn resulted in an increase in the substrate specificity of E.coli PQQGDH (Sode and Kojima, 1997). As shown in Table III, all the mutants containing the His substitution to Asn gained increased substrate specificity. Therefore, this substitution is effective in improving the substrate specificity even if other substitutions are simultaneously introduced, such as the region responsible for EDTA tolerance and for the increase in thermal stability. The other remarkable feature of His substitution to Asn, shown in Figure 5a, was that the substitution resulted in the decrease in the thermal stability. As observed in the all β-propeller protein, the final W-motif (W8 motif in the case of PQQGDH) is composed of the N-terminal region and the C-terminal region which includes the A3 region (Figure 7). The final W8 motif consists of four β-strands (W8a, W8b, W8c, W8d); three β-strands (W8a, W8b, W8c) which are from the C-terminal region, and the rest (W8d) is from the Nterminal region. The construction of the final motif by forming hydrogen bonds between W8c and W8d are peculiar to β-propeller protein. Consequently, the W8 motif may interact with the W1 motif, which is composed of the N-terminal region, to construct a β-propeller structure. Therefore, the conformational stability of W8 motif of PQQGDH may play a significant role in the whole PQQGDH conformational stability. Since His775 is involved in the W8 motif in the predicted model, it is supposed that substitution of His775 to Asn resulted in the imbalance of the interaction within W8 69

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and/or between the N-terminal region and C-terminal region. Besides, as the A3 region at the C-terminal end is also involved in the interaction with the N-terminal region (Figure 7), it subsequently had a significant effect on thermal stability. Although the study on the mechanisms of stabilization by the introduction of the A3 region is still in progress, the contribution of the A3 region towards thermal stability complemented the decrease in the thermal stability due to the His775 to Asn substitution. The chimeric PQQGDH which harbours all the regions in the target, E32A27E38A3 H782N, was constructed and it was revealed that this chimeric enzyme retains all of the improved enzymatic properties. Although the catalytic activity judged from the Vmax/Km value of E32A27E38A3 H782N decreased upon introduction of the A27 region, this enzyme could be potentially advantageous in the application for a glucose enzyme sensor component compared with the wild-type enzyme. In the blood glucose measurement system, the target samples, particularly the samples from the diabetes patients, contain more than 200 mg dl–1 (about 11 mM) glucose. If these target samples contain such a high glucose concentration, the utilization of an enzyme showing both the high Km and Vmax values is ideal for enzyme sensor construction, particularly for the measurement of whole blood samples without dilution. Although the Vmax value of E32A27E38A3 H782N for glucose is about 24% that of wild-type PQQGDH, the increased Km value (about 5-fold) complements the decreased Vmax value. Moreover, this engineered enzyme gained increased thermal stability, EDTA tolerance and narrowed substrate specificity, and the enzyme we constructed had obviously improved its properties considering its application in enzyme sensor construction. Acknowledgements This research was partly supported by Grant-in-Aid for Priority Area Research no. 10145106 from the Ministry of Education, Science, Sports and Culture of Japan 1998.

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