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Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase
Enzyme and Microbial Technology 44 (2009) 52–56
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase Seungsu Kim, Seiji Miura, Stefano Ferri, Wakako Tsugawa, Koji Sode ∗ Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 17 June 2008 Received in revised form 1 September 2008 Accepted 2 September 2008 Keywords: Biosensor Diabetes Fructosyl amine oxidase Hemoglobin A1c (HbA1c ) Substrate specificity
a b s t r a c t Site-directed mutagenesis was carried out at the putative active site of fructosyl amine oxidase (FAOD) to improve its substrate specificity based on information from the structural model. Substitution of the His51 residue with other amino acids predicted to interact with the substrate resulted in mutant FAODs with improved specificity for fructosyl-˛ N-valine (f-˛ Val), a model compound of hemoglobin A1c (HbA1c ). Kinetic analysis of these mutant FAODs indicated that these His51 variants had decreased Vmax Km −1 values for fructosyl-ε N-lysine (f-ε Lys) compared to the wild-type enzyme, while the Vmax Km −1 values for f-˛ Val remained unaffected or were increased. Among the 19 variants at His51, His51Lys/Arg was combined with previously reported mutants, such as Asn354His [Miura S, Ferri S, Tsugawa W, Kim S, Sode K. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel 2008;21:233–9]; the His51/Asn354 double mutant showed a greater improvement in the specificity for f-˛ Val over f-ε Lys and higher activity toward f-˛ Val than the single mutants and the wild-type. In order to develop a biosensor for the measurement of HbA1c , an FAOD enzyme specific to f-␣ Val is required to avoid influence of f- Lys derived from other glycated proteins. Our results support the proposed 3D model, and the resulting f-␣ Val-specific mutants are expected to be applied to the enzymatic measurement of HbA1c . © 2008 Elsevier Inc. All rights reserved.
1. Introduction Glycation involves a series of non-enzymatic reactions between reducing sugars, such as glucose, and the free amino groups in amino acids or proteins. Since glycation also occurs in blood proteins, the levels of glycated blood proteins, particularly glycated hemoglobin (hemoglobin A1c , HbA1c ) and glycated albumin, reflect the past mean blood glucose concentration. Because HbA1c and glycated albumin are recognized as important markers in the diagnosis and glycemic control of diabetes mellitus [2–4], there is a considerable importance placed on the measurement of these glycated proteins. A number of methods are available for the determination of glycated compounds, such as HPLC, immunoassay and mass spectrometry. However, because these methods are generally complicated and expensive, there remains a strong demand for a simple and economical sensor system that can be applied for the point-ofcare or home monitoring of diabetes patients. The development of an enzyme-based sensor employing the enzyme fructosyl amine oxidase (FAOD), or amadoriase, is an approach with a great potential for satisfying such a demand. FAODs are the enzymes catalyzing the oxidative deglycation of fructosyl
∗ Corresponding author. Fax: +81 42 388 7027. E-mail address: [email protected] (K. Sode). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.09.001
amino acids to produce glucosone, the amino acid and hydrogen peroxide. FAODs have been isolated from several different microorganisms, including filamentous fungi [5–10], a marine yeast [11] and bacteria [12,13], and they have been characterized. We have previously isolated an FAOD from the marine yeast strain Pichia sp. N1-1, and have succeeded in the cloning of the N1-1 FAOD gene and its recombinant production in Escherichia coli [14]. We have also developed FAOD-based biosensor systems capable of effectively measuring fructosyl-␣ N-valine (f-␣ Val) [15–17]. HbA1c results from the glycation of the N-terminal valine residue of the hemoglobin  subunit. HbA1c is a major glycemic indicator for diabetes patients, where its level reflects the average blood glucose concentration over the past 1–2 months. Glycated albumin, which results from the glycation of the amino group on the side chain of internal lysine residues, has also become an important diabetes indicator. Because the FAODs reported so far are unable to oxidize glycated proteins directly, the enzymatic measurement of HbA1c or glycated albumin must be preceded by a proteolytic digestion step to liberate f-␣ Val or fructosyl- N-lysine (f- Lys), respectively. A biosensor for the measurement of either HbA1c or glycated albumin would ideally employ an FAOD enzyme specific to the corresponding glycated amino acid. Because wild-type N11 FAOD exhibits enzymatic activity toward both f-␣ Val and f- Lys, the engineering of this enzyme is required for its application to the measurement of specific glycated proteins. Extensive international
S. Kim et al. / Enzyme and Microbial Technology 44 (2009) 52–56
studies have demonstrated a clear correlation between HbA1c and the development of long-term complications from diabetes, and have recognized HbA1c as the gold standard for assessing glycemic control [18,19]. This encouraged us to develop an enzymatic biosensor system for the measurement of HbA1c . We have recently proposed a 3D structure model of N1-1 FAOD, identifying the residues located around the putative active site [20]. Site-directed mutagenesis and docking models produced using the N1-1 FAOD structural model with the substrates revealed that Asn354 plays different roles in the recognition of f-␣ Val and f- Lys [1]. Substitution of Asn354, which interacts closely with f- Lys, but not with f-␣ Val, with histidine and lysine resulted in a considerable improvement of the substrate specificity for f-␣ Val. In our previous study [1], we found that the substrate specificity of both the wild-type and the Asn354 mutant was affected by the buffer concentration. Whereas the Asn354 mutant showed high specificity and activity toward f-␣ Val at a potassium phosphate buffer (PPB) concentration of 500 mM, the activity toward f- Lys of this Asn354 mutant FAOD was still not negligible at low (5 mM) PPB concentration. Since such a high (500 mM) buffer concentration is not suitable for practical diagnostic application, it is still required for N1-1 FAOD to be specific to f-␣ Val regardless of the buffer concentration in order for it to be applicable to the measurement of HbA1c . Our recent success in the improvement of the substrate specificity of N1-1 FAOD using our original structural information encouraged us to take a rational design approach. In this study, we carried out site-directed mutagenesis at His51, a residue that was predicted to recognize the carboxyl group in substrates. Based on the results, we created the His51Lys/Asn354His and His51Arg/Asn354His mutants, which showed improved specificity for f-␣ Val and high activity toward f-␣ Val. These mutant FAODs are expected to be applied to the enzymatic determination of HbA1c . 2. Materials and methods
53
Table 1 The sequences of the oligonucleotide primers used for the site-directed mutagenesis of N1-1 FAOD His51Ala His51Cys His51Asp His51Glu His51Phe His51Gly His51Ile His51Lys His51Leu His51Met His51Asn His51Pro His51Gln His51Arg His51Ser His51Thr His51Val His51Trp His51Tyr Asn354Lys Asn354His
5 -GTAACAAGATTTTTGCGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGATTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGAATACGATTATGTTGC-3 5 -GTAACAAGATTTTTTTTTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGGTTACGATTATGTTGC-3 5 -GTAACAAGATTTTTATCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAAATACGATTATGTTGC-3 5 -GTAACAAGATTTTTCTGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTATGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAACTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCCGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCAGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTACCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGTGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTGGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTATTACGATTATGTTGC-3 5 -CGGGTGACTCTGGAAAATCGTTCAAGATC-3 5 -CGGGTGACTCTGGACACTCGTTCAAGATC-3
The mutation sites are indicated by bold underlined letters.
(GE Healthcare UK, Ltd., Buckinghamshire, England) equilibrated with 10 mM PPB, pH 8.0. The absorbed protein was eluted with a 0–0.25 M linear NaCl gradient. The active fractions were pooled, and flavin adenine dinucleotide (FAD) was added to a final concentration of 100 M. Ammonium sulfate was added to 35% saturation, and the precipitated protein was collected by centrifugation at 15,000 × g for 20 min. The supernatant was dialyzed at 4 ◦ C against 10 mM PPB, pH 8.0, containing 100 M FAD and 1% mannose, and subsequently dialyzed against 10 mM PPB, pH 8.0, containing 100 M FAD. The dialyzed enzyme solution was applied to a RESOURCE Q column equilibrated with 10 mM PPB, pH 8.0. The absorbed protein was eluted with a linear NaCl gradient (0.0–0.10 M) in the same buffer. The active fractions were collected, and FAD was added to a final concentration of 100 M. Finally, the purified enzyme solution was dialyzed against 10 mM PPB, pH 7.0, and stored at 4 ◦ C. The purity of the purified enzymes was confirmed by SDS-PAGE (data not shown), and their concentrations were measured using a DC Protein Assay Kit (Bio-Rad, CA, USA).
2.1. Materials 2.4. Enzyme assay F-␣ Val and f- Lys were prepared according to the method of Keil et al. [21]. 4-Aminoantipyrine was obtained from Kanto Kagaku (Tokyo, Japan). N,NBis(4-sulfobutyl)-3-methylaniline disodium salt (TODB) were obtained from Dojin (Kumamoto, Japan). Horseradish peroxidase was obtained from Amano Enzymes (Nagoya, Japan). All other chemicals were of reagent grade. 2.2. Site-directed mutagenesis The FAOD expression vector pTN1 was previously constructed based on pTrc99A [1]. The FAOD expression vector pETN1 was constructed by inserting the N1-1 FAOD gene into pET-28(a) (Merck Biosciences, Darmstadt, Germany). Site-directed mutagenesis was carried out using the QuikChange® mutagenesis kit (Stratagene, CA, USA) in accordance with the manufacturer’s instructions using the oligonucleotides summarized in Table 1 and their respective complementary oligonucleotides. The mutations were confirmed by sequencing with an ABI Prism BigDye Terminator cycle sequencing kit v3.0 on an ABI Prism 3100 Genetic Analyzer (Applied Biosytems, CA, USA). 2.3. Expression and preparation of wild-type and mutant FAODs The FAOD expression vector pTN1 was used for the preparation of crude His51 single-mutant enzymes (i), and pETN1 was used for the preparation of cell extracts and the purification of some His51 single-mutant and His51/Asn354 double-mutant enzymes (ii). E. coli DH5␣ transformed with the original wild-type or mutated pTN1 (i) or BL21(DE3) transformed with the original wild-type or mutated pETN1 (ii) were grown aerobically at 37 ◦ C in LB medium containing 100 g ampicillin ml−1 (i) or 50 g kanamycin ml−1 (ii). After reaching an A660 nm value of 0.6, the cells were induced with 0.3 mM (i) or 0.4 mM (ii) isopropyl--d-thiogalactopyranoside (IPTG), and the incubation was continued at 30 ◦ C for 6 h (i) or 25 ◦ C for 4 h (ii). The cells were harvested by centrifugation and resuspended in 10 mM PPB, pH 7.0, and lysed by sonication (i) or by 5 passages through a French press (1500 kg cm−2 ) (ii). The lysate was centrifuged at 10,000 × g at 4 ◦ C for 20 min, and the supernatant was centrifuged at 177,100 × g at 4 ◦ C for 90 min. The supernatant was then dialyzed against 10 mM PPB, pH 8.0, and the crude enzyme solution was assayed (i) or further purified as follows (ii) [1,20]. The crude FAOD was applied to a RESOURCE Q column
The FAOD activity was assayed at 25 ◦ C in 5 or 500 mM PPB, pH 7.0, 1.5 mM TODB, 2 U ml−1 horseradish peroxidase, and 1.5 mM 4-aminoantipyrine in the presence of substrates at various concentrations [14]. The formation of quinoneimine dye was measured spectrophotometrically at 546 nm. One unit was defined as the enzyme quantity that oxidizes 1 mole of substrate per minute under the above reaction conditions.
3. Results and discussion 3.1. Proposal of mutation residue from structural studies and comparison with other FAODs Based on the previously proposed 3D structure model of N1-1 FAOD and enzyme–substrate docking models, several amino acid residues around the putative active site region, which are likely to play important roles in the enzymatic reaction and substrate recognition were identified [20]. His51 was identified as a residue involved in the recognition of the carboxyl groups of substrates [1,20]. Most of the other residues located around the putative active site of the structural model of N1-1 FAOD were relatively wellconserved in eukaryotic FAODs; however, His51 was not conserved (Fig. 1), and neither was Asn354 [1]. The eukaryotic FAODs can be classified into three groups based on their substrate specificities: FAODs showing a high preference for (i) ␣-glycated, (ii) -glycated amino acids, and (iii) FAODs able to oxidize ␣- and -glycated amino acids at comparable levels (Fig. 1). Interestingly, only the FAODs of the first group, i.e., ␣-fructosyl amino acid-specific FAODs, had a histidine residue at the position corresponding to the Asn354 of N1-1 FAOD, and this was consistent with the improvement of specificity
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S. Kim et al. / Enzyme and Microbial Technology 44 (2009) 52–56
Fig. 1. Alignment of the amino acid sequence around the His51 of N1-1 FAOD with other eukaryotic FAODs. The amino acid positions of the sequences are shown in parentheses. The enzymes are grouped according to substrate specificity: (i) preference for ␣-fructosyl amino acids, (ii) preference for -fructosyl amino acids, and (iii) similar activity with both ␣- and -glycated amino acids. The sequences are those of FAOD-P from Penicillum janthinelum AKU 3413, FAOD-U from Ulocladium sp. JS-103, FPOX-E from Eupenicillum terrenum ATCC 18547, FPOX-C from Coniochaeta sp. NISL 9330, FAOD-F from Fusarium oxysporum NBRC 9972, FAOD-A from Aspergillus terreus GP1, Amadoriases I from Aspergillus fumigatus, FAOD-Ao1 from Aspergillus oryzae, Amadoriases II from Aspergillus fumigatus, FAOD-Ao2 from Aspergillus oryzae and N1-1 FAOD from Pichia sp. N1-1. The arrowhead indicates the residues corresponding to the His51 of N1-1 FAOD. Identical amino acids are highlighted in black or dark gray and conserved amino acids in light gray.
for f-␣ Val observed in the Asn354His mutant FAOD which we created recently [1]. However, no histidine or positively charged amino acid was observed at the position corresponding to the His51 of N1-1 FAOD in any other FAODs, and there was also no particular amino acid conservation at this position among the FAODs of each group (Fig. 1). This led us to modify His51 in order to investigate the role of this residue on the enzymatic reaction, and it was expected that His51 mutants with various enzymatic properties would be obtained. 3.2. Site-directed mutagenesis of His51 We carried out the amino acid substitution of His51 with 19 other amino acids. As shown in Table 2, most of the created mutants showed no significant change in substrate specificity and decreased activity toward f-␣ Val, except for the substitutions with Lys, Arg, Trp and Tyr. His51Lys showed higher activity toward f-␣ Val compared to the wild-type and decreased activity toward f- Lys; i.e., it showed a slightly improved substrate specificity for f-␣ Val. Table 2 Activities and substrate specificities of crude wild-type and His51 mutant FAODs Activitya (mU mg−1 )
Wild-type His51Lys His51Arg His51Trp His51Tyr His51Ala His51Leu His51Ile His51Val His51Phe His51Cys His51Met His51Asn His51Gln His51Ser His51Thr His51Asp His51Glu His51Gly His51Pro
f-˛ Val
f-ε Lys
110 (100%) 170 (152%) 79 (72%) 37 (34%) 62 (56%) 7.2 (7%) 27 (24%) 17 (15%) 41 (38%) 50 (46%) 33 (30%) 23 (21%) 1.0 (1%) 10 (10%) 3.2 (3%) 14 (13%) 3.2 (3%) 1.1 (1%) 0.20 (0%) n.d.
340 (100%) 280 (80%) 24 (7%) 36 (10%) 61 (18%) 59 (17%) 110 (33%) 90 (26%) 140 (40%) 77 (22%) 200 (57%) 74 (22%) 89 (26%) 23 (7%) 64 (19%) 99 (29%) 4.5 (1%) 9.4 (3%) 9.1 (3%) n.d.
Activity for f-ε Lys/activity for f-˛ Val
3.2 1.5 0.31 0.96 0.99 8.2 4.2 5.4 3.4 1.5 5.9 3.3 88 2.2 20 7.2 1.4 8.3 45 –
The activities which could not be detected are represented as “n.d.”. a The activity was determined with 1 mM f-˛ Val or f-ε Lys in 5 mM PPB.
His51Arg showed improved substrate specificity for f-␣ Val (activity for f- Lys/activity for f-␣ Val: 0.31) as compared with the wild-type (3.2), maintaining relatively high activity toward f-␣ Val. His51Trp and His51Tyr both showed improved substrate specificity for f-␣ Val (0.96 for His51Trp and 0.99 for His51Tyr); however, their activities toward f-␣ Val markedly decreased. His51 was predicted to recognize the carboxyl group of substrates in the N1-1 FAOD substrate-binding model [20]. It was expected that several mutant FAODs with various properties would be obtained by introducing mutations at His51, according to the nature of the new amino acid. Substitutions with Gly, Ser, or Glu resulted in markedly decreased activities toward both substrates, although other eukaryotic FAODs have theses amino acids at the position corresponding to His51 of N1-1 FAOD. No significant amino acid sequence similarities in this region (His51 to Arg66 in N1-1 FAOD, Fig. 1) suggests that the role of His51 in N1-1 FAOD may be unique. Since the substitution with acidic amino acids (Asp and Glu) or a small amino acid (Gly) resulted in less than 5% of the wild-type activity toward both substrates, it is supposed that the interaction between His51 and the carboxyl groups in substrates is crucial for the activity of this FAOD. On the other hand, mutants, which were created by substituting His51 with amino acids having a basic side chain, His51Lys and His51Arg, retained the high activity toward f-␣ Val. This result suggests that electrostatic interaction plays a crucial role in the recognition of f-␣ Val. Mutations with hydrophobic amino acids showed activities (>15%) toward both substrates. It was considered that the hydrophobicity of the active site may also be important for the recognition of the substrates. However, alteration to bulky residues (Arg, Trp, Phe) resulted in markedly decreased activities toward f- Lys compared with f-␣ Val. According to our enzyme–substrate docking model, the predicted distance between His51 and the carboxyl group of f-␣ Val is longer than that between His51 and the carboxyl group of f- Lys [1]. Steric hindrance between f- Lys and the bulky side chains may be elicited by the mutations, and it was suggested that appropriate bulkiness of the amino acids in this position is important for the interaction with the substrate and may affect the substrate specificity. Since His51Lys and His51Arg showed improved activity and substrate specificity toward f-␣ Val, respectively, we further purified and characterized these two His51 mutant FAODs. As shown in Table 3, His51Lys showed an increased Vmax Km −1 value for f-˛ Val, 4.2 U mg−1 mM−1 , compared to the wild-type (1.7 U mg−1 mM−1 ). The Vmax Km −1 value for f-ε Lys of His51Lys,
S. Kim et al. / Enzyme and Microbial Technology 44 (2009) 52–56
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Table 3 Kinetic parameters of purified wild-type and mutant FAODs f-˛ Val
Wild-type His51Lys His51Arg His51Lys/Asn354His His51Arg/Asn354His Asn354His [1] Asn354Lys [1] a
f-ε Lys
Km (mM)
Vmax (U mg
3.9 2.4 11 0.4 2.4 0.6 2.0
6.5 10 17 11 15 16 25
−1
)
Vmax Km
−1
−1
(A) (U mg
−1
mM
1.7 4.2 1.5 28 6.3 27 13
)
B/A
Km (mM)
Vmax (U mg
0.9 2.0 18 27 –a 3.9 15
9.5 15 8.5 33 –a 16 21
−1
)
Vmax Km 11 7.5 0.47 1.2 –a 4.1 1.4
−1
−1
(B) (U mg
−1
mM
) 6.5 1.8 0.31 0.043 –a 0.15 0.11
Not determined.
7.5 U mg−1 mM−1 , was slightly lower than the 11 U mg−1 mM−1 of the wild-type; this means that the substrate specificity (Vmax Km −1 for f- Lys/Vmax Km −1 for f-˛ Val) was improved about 3.6-fold (1.8 for His51Lys, 6.5 for the wild-type). His51Arg retained the Vmax Km −1 value for f-˛ Val, 1.5 U mg−1 mM−1 , and had a greatly decreased Vmax Km −1 value for f-ε Lys, 0.5 U mg−1 mM−1 , compared to the wild-type; this indicates that the substrate specificity was drastically improved, by about 21 times (0.31 for His51Arg). Lys and Arg are basic amino acids with relatively bulky side chains. The positive charge and the appropriate bulkiness of the side chain of Lys lent His51Lys a considerably enhanced activity toward f-˛ Val and a slightly decreased activity toward f-ε Lys. In the case of His51Arg, steric hindrance may have played a particularly strong role in the interaction between f-ε Lys and Arg; this resulted in a remarkable decrease in the mutant enzyme’s activity toward f-ε Lys. 3.3. Characterization of His51Lys/Asn354His and His51Arg/Asn354His We then combined His51Lys and His51Arg with the previously reported Asn354His mutation [1] to improve the substrate specificity. As shown in Table 3, His51Lys/Asn354His showed a greatly increased Vmax Km −1 value for f-˛ Val, 28 U mg−1 mM−1 , and a greatly decreased Vmax Km −1 value for f-ε Lys, 1.2 U mg−1 mM−1 ; this means that the substrate specificity was improved about 125fold (0.043). His51Arg/Asn354His showed a 3.7-fold increase in the Vmax Km −1 value for f-˛ Val, 6.3 U mg−1 mM−1 , compared with the wild-type, and had almost no activity toward f-ε Lys. By combining the two amino acid substitutions, His51Arg and Asn354His, we succeeded in constructing an engineered FAOD, which has the cumulative effect of each single mutation. These two double-mutant FAODs showed a greater improvement in specificity for f-˛ Val over f-ε Lys than the wild-type and the single mutants even at low (5 mM) PPB concentration. The resulting double mutants are expected to be applied to the practical enzymatic measurement of HbA1c . Recently, the improvement of the substrate specificity of FAOD from Ulocladium sp. (FAOD-U) and Fusarium oxysporum (FAOD-F) has been reported [22,23]. However, these achievements depended on the random mutagenesis approach, and the substrate specificities toward their original preferred substrates (f-␣ Val for wild-type FAOD-U, f- Lys for wild-type FAOD-F) were improved. Furthermore, the discussion about the effect of the resulting mutation was not sufficient, because there was no information on the putative active site or the 3D structure. In this study, the constructed double mutants showed high specificity toward f-␣ Val, while wildtype N1-1 FAOD showed higher activity toward f- Lys than f-␣ Val. Furthermore, it is considered that our achievement is a result of a rational design approach based on the information of the proposed original structural model.
The mutant FAODs His51Lys/Asn354His and His51Arg/ Asn354His showed improved substrate specificity toward f␣ Val, which means that they have a great potential for use in the measurement of HbA1c in whole blood, because the interference from glycated albumin would be greatly reduced. In addition to creating mutant FAODs that are expected to be applied in the measurement of HbA1c , we have succeeded in combining two mutations in order to produce double mutants with a cumulative effect of the mutations. We demonstrated that our original structural model was useful for the engineering of N1-1 FAOD, and it is expected that various mutant FAODs with improved properties, such as peptidase resistance and activity, will be created based on this information. Furthermore, various mutations can be combined to create an ideal FAOD, and this approach will contribute to the development of enzyme-based sensing systems for glycated proteins. References [1] Miura S, Ferri S, Tsugawa W, Kim S, Sode K. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel 2008;21:233–9. [2] Klenk DC, Hermanson GT, Krohn RI, Fujimoto EK, Mallia AK, Smith PK, et al. Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences. Clin Chem 1982;28:2088–94. [3] Johnson RN, Metcalf PA, Baker JR. Fructosamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin Chim Acta 1983;127:87–95. [4] Iberg N, Flückiger R. Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites. J Biol Chem 1986;261:13542–5. [5] Yoshida N, Sakai Y, Serata M, Tani Y, Kato N. Distribution and properties of fructosyl amino acid oxidase in fungi. Appl Environ Microbiol 1995;61: 4487–9. [6] Yoshida N, Sakai Y, Isogai A, Fukuya H, Yagi M, Tani Y, et al. Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins. Eur J Biochem 1996;242:499–505. [7] Takahashi M, Pischetsrieder M, Monnier VM. Isolation, purification, and characterization of amadoriase isoenzymes (fructosyl amine-oxygen oxidoreductase (EC 1.5.3) from Aspergillus sp. J Biol Chem 1997;272:3437–43. [8] Takahashi M, Pischetsrieder M, Monnier VM. Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine-oxygen oxidoreductase, (EC1.5.3) from Aspergillus fumigates. J Biol Chem 1997;272:12505–7. [9] Hirokawa K, Gomi K, Bakke M, Kajiyama N. Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi. Arch Microbiol 2003;180: 227–31. [10] Akazawa S, Karino T, Yoshida N, Katsuragi T, Tani Y. Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae. Appl Environ Microbiol 2004;70:5882–90. [11] Sode K, Ishimura F, Tsugawa W. Screening and characterization of fructosyl– valine-utilizing marine microorganisms. Mar Biotechnol 2001;3:126–32. [12] Horiuchi T, Kurokawa T, Saito N. Purification and properties of fructosyl amino acid oxidase from Corynebacterium sp. 2-4-1. Agric Biol Chem 1989;53:103–10. [13] Ferri S, Sakaguchi A, Goto H, Tsugawa W, Sode K. Isolation and characterization of fructosyl-amine oxidase from an Arthrobacter sp. Biotechnol Lett 2005;27:27–32. [14] Ferri S, Miura S, Sakaguchi A, Ishimura F, Tsugawa W, Sode K. Cloning and expression of fructosyl-amine oxidase from marine yeast Pichia sp. N1-1. Mar Biotechnol 2004;6:625–32. [15] Tsugawa W, Ishimura F, Ogawa K, Sode K. Development of an enzyme sensor utilizing a novel fructosyl amine oxidase from a marine yeast. Electrochemistry 2000;68:869–71.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase Seungsu Kim, Seiji Miura, Stefano Ferri, Wakako Tsugawa, Koji Sode ∗ Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 17 June 2008 Received in revised form 1 September 2008 Accepted 2 September 2008 Keywords: Biosensor Diabetes Fructosyl amine oxidase Hemoglobin A1c (HbA1c ) Substrate specificity
a b s t r a c t Site-directed mutagenesis was carried out at the putative active site of fructosyl amine oxidase (FAOD) to improve its substrate specificity based on information from the structural model. Substitution of the His51 residue with other amino acids predicted to interact with the substrate resulted in mutant FAODs with improved specificity for fructosyl-˛ N-valine (f-˛ Val), a model compound of hemoglobin A1c (HbA1c ). Kinetic analysis of these mutant FAODs indicated that these His51 variants had decreased Vmax Km −1 values for fructosyl-ε N-lysine (f-ε Lys) compared to the wild-type enzyme, while the Vmax Km −1 values for f-˛ Val remained unaffected or were increased. Among the 19 variants at His51, His51Lys/Arg was combined with previously reported mutants, such as Asn354His [Miura S, Ferri S, Tsugawa W, Kim S, Sode K. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel 2008;21:233–9]; the His51/Asn354 double mutant showed a greater improvement in the specificity for f-˛ Val over f-ε Lys and higher activity toward f-˛ Val than the single mutants and the wild-type. In order to develop a biosensor for the measurement of HbA1c , an FAOD enzyme specific to f-␣ Val is required to avoid influence of f- Lys derived from other glycated proteins. Our results support the proposed 3D model, and the resulting f-␣ Val-specific mutants are expected to be applied to the enzymatic measurement of HbA1c . © 2008 Elsevier Inc. All rights reserved.
1. Introduction Glycation involves a series of non-enzymatic reactions between reducing sugars, such as glucose, and the free amino groups in amino acids or proteins. Since glycation also occurs in blood proteins, the levels of glycated blood proteins, particularly glycated hemoglobin (hemoglobin A1c , HbA1c ) and glycated albumin, reflect the past mean blood glucose concentration. Because HbA1c and glycated albumin are recognized as important markers in the diagnosis and glycemic control of diabetes mellitus [2–4], there is a considerable importance placed on the measurement of these glycated proteins. A number of methods are available for the determination of glycated compounds, such as HPLC, immunoassay and mass spectrometry. However, because these methods are generally complicated and expensive, there remains a strong demand for a simple and economical sensor system that can be applied for the point-ofcare or home monitoring of diabetes patients. The development of an enzyme-based sensor employing the enzyme fructosyl amine oxidase (FAOD), or amadoriase, is an approach with a great potential for satisfying such a demand. FAODs are the enzymes catalyzing the oxidative deglycation of fructosyl
∗ Corresponding author. Fax: +81 42 388 7027. E-mail address: [email protected] (K. Sode). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.09.001
amino acids to produce glucosone, the amino acid and hydrogen peroxide. FAODs have been isolated from several different microorganisms, including filamentous fungi [5–10], a marine yeast [11] and bacteria [12,13], and they have been characterized. We have previously isolated an FAOD from the marine yeast strain Pichia sp. N1-1, and have succeeded in the cloning of the N1-1 FAOD gene and its recombinant production in Escherichia coli [14]. We have also developed FAOD-based biosensor systems capable of effectively measuring fructosyl-␣ N-valine (f-␣ Val) [15–17]. HbA1c results from the glycation of the N-terminal valine residue of the hemoglobin  subunit. HbA1c is a major glycemic indicator for diabetes patients, where its level reflects the average blood glucose concentration over the past 1–2 months. Glycated albumin, which results from the glycation of the amino group on the side chain of internal lysine residues, has also become an important diabetes indicator. Because the FAODs reported so far are unable to oxidize glycated proteins directly, the enzymatic measurement of HbA1c or glycated albumin must be preceded by a proteolytic digestion step to liberate f-␣ Val or fructosyl- N-lysine (f- Lys), respectively. A biosensor for the measurement of either HbA1c or glycated albumin would ideally employ an FAOD enzyme specific to the corresponding glycated amino acid. Because wild-type N11 FAOD exhibits enzymatic activity toward both f-␣ Val and f- Lys, the engineering of this enzyme is required for its application to the measurement of specific glycated proteins. Extensive international
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studies have demonstrated a clear correlation between HbA1c and the development of long-term complications from diabetes, and have recognized HbA1c as the gold standard for assessing glycemic control [18,19]. This encouraged us to develop an enzymatic biosensor system for the measurement of HbA1c . We have recently proposed a 3D structure model of N1-1 FAOD, identifying the residues located around the putative active site [20]. Site-directed mutagenesis and docking models produced using the N1-1 FAOD structural model with the substrates revealed that Asn354 plays different roles in the recognition of f-␣ Val and f- Lys [1]. Substitution of Asn354, which interacts closely with f- Lys, but not with f-␣ Val, with histidine and lysine resulted in a considerable improvement of the substrate specificity for f-␣ Val. In our previous study [1], we found that the substrate specificity of both the wild-type and the Asn354 mutant was affected by the buffer concentration. Whereas the Asn354 mutant showed high specificity and activity toward f-␣ Val at a potassium phosphate buffer (PPB) concentration of 500 mM, the activity toward f- Lys of this Asn354 mutant FAOD was still not negligible at low (5 mM) PPB concentration. Since such a high (500 mM) buffer concentration is not suitable for practical diagnostic application, it is still required for N1-1 FAOD to be specific to f-␣ Val regardless of the buffer concentration in order for it to be applicable to the measurement of HbA1c . Our recent success in the improvement of the substrate specificity of N1-1 FAOD using our original structural information encouraged us to take a rational design approach. In this study, we carried out site-directed mutagenesis at His51, a residue that was predicted to recognize the carboxyl group in substrates. Based on the results, we created the His51Lys/Asn354His and His51Arg/Asn354His mutants, which showed improved specificity for f-␣ Val and high activity toward f-␣ Val. These mutant FAODs are expected to be applied to the enzymatic determination of HbA1c . 2. Materials and methods
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Table 1 The sequences of the oligonucleotide primers used for the site-directed mutagenesis of N1-1 FAOD His51Ala His51Cys His51Asp His51Glu His51Phe His51Gly His51Ile His51Lys His51Leu His51Met His51Asn His51Pro His51Gln His51Arg His51Ser His51Thr His51Val His51Trp His51Tyr Asn354Lys Asn354His
5 -GTAACAAGATTTTTGCGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGATTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGAATACGATTATGTTGC-3 5 -GTAACAAGATTTTTTTTTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGGTTACGATTATGTTGC-3 5 -GTAACAAGATTTTTATCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAAATACGATTATGTTGC-3 5 -GTAACAAGATTTTTCTGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTATGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAACTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCCGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCAGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTCGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTAGCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTACCTACGATTATGTTGC-3 5 -GTAACAAGATTTTTGTGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTGGTACGATTATGTTGC-3 5 -GTAACAAGATTTTTTATTACGATTATGTTGC-3 5 -CGGGTGACTCTGGAAAATCGTTCAAGATC-3 5 -CGGGTGACTCTGGACACTCGTTCAAGATC-3
The mutation sites are indicated by bold underlined letters.
(GE Healthcare UK, Ltd., Buckinghamshire, England) equilibrated with 10 mM PPB, pH 8.0. The absorbed protein was eluted with a 0–0.25 M linear NaCl gradient. The active fractions were pooled, and flavin adenine dinucleotide (FAD) was added to a final concentration of 100 M. Ammonium sulfate was added to 35% saturation, and the precipitated protein was collected by centrifugation at 15,000 × g for 20 min. The supernatant was dialyzed at 4 ◦ C against 10 mM PPB, pH 8.0, containing 100 M FAD and 1% mannose, and subsequently dialyzed against 10 mM PPB, pH 8.0, containing 100 M FAD. The dialyzed enzyme solution was applied to a RESOURCE Q column equilibrated with 10 mM PPB, pH 8.0. The absorbed protein was eluted with a linear NaCl gradient (0.0–0.10 M) in the same buffer. The active fractions were collected, and FAD was added to a final concentration of 100 M. Finally, the purified enzyme solution was dialyzed against 10 mM PPB, pH 7.0, and stored at 4 ◦ C. The purity of the purified enzymes was confirmed by SDS-PAGE (data not shown), and their concentrations were measured using a DC Protein Assay Kit (Bio-Rad, CA, USA).
2.1. Materials 2.4. Enzyme assay F-␣ Val and f- Lys were prepared according to the method of Keil et al. [21]. 4-Aminoantipyrine was obtained from Kanto Kagaku (Tokyo, Japan). N,NBis(4-sulfobutyl)-3-methylaniline disodium salt (TODB) were obtained from Dojin (Kumamoto, Japan). Horseradish peroxidase was obtained from Amano Enzymes (Nagoya, Japan). All other chemicals were of reagent grade. 2.2. Site-directed mutagenesis The FAOD expression vector pTN1 was previously constructed based on pTrc99A [1]. The FAOD expression vector pETN1 was constructed by inserting the N1-1 FAOD gene into pET-28(a) (Merck Biosciences, Darmstadt, Germany). Site-directed mutagenesis was carried out using the QuikChange® mutagenesis kit (Stratagene, CA, USA) in accordance with the manufacturer’s instructions using the oligonucleotides summarized in Table 1 and their respective complementary oligonucleotides. The mutations were confirmed by sequencing with an ABI Prism BigDye Terminator cycle sequencing kit v3.0 on an ABI Prism 3100 Genetic Analyzer (Applied Biosytems, CA, USA). 2.3. Expression and preparation of wild-type and mutant FAODs The FAOD expression vector pTN1 was used for the preparation of crude His51 single-mutant enzymes (i), and pETN1 was used for the preparation of cell extracts and the purification of some His51 single-mutant and His51/Asn354 double-mutant enzymes (ii). E. coli DH5␣ transformed with the original wild-type or mutated pTN1 (i) or BL21(DE3) transformed with the original wild-type or mutated pETN1 (ii) were grown aerobically at 37 ◦ C in LB medium containing 100 g ampicillin ml−1 (i) or 50 g kanamycin ml−1 (ii). After reaching an A660 nm value of 0.6, the cells were induced with 0.3 mM (i) or 0.4 mM (ii) isopropyl--d-thiogalactopyranoside (IPTG), and the incubation was continued at 30 ◦ C for 6 h (i) or 25 ◦ C for 4 h (ii). The cells were harvested by centrifugation and resuspended in 10 mM PPB, pH 7.0, and lysed by sonication (i) or by 5 passages through a French press (1500 kg cm−2 ) (ii). The lysate was centrifuged at 10,000 × g at 4 ◦ C for 20 min, and the supernatant was centrifuged at 177,100 × g at 4 ◦ C for 90 min. The supernatant was then dialyzed against 10 mM PPB, pH 8.0, and the crude enzyme solution was assayed (i) or further purified as follows (ii) [1,20]. The crude FAOD was applied to a RESOURCE Q column
The FAOD activity was assayed at 25 ◦ C in 5 or 500 mM PPB, pH 7.0, 1.5 mM TODB, 2 U ml−1 horseradish peroxidase, and 1.5 mM 4-aminoantipyrine in the presence of substrates at various concentrations [14]. The formation of quinoneimine dye was measured spectrophotometrically at 546 nm. One unit was defined as the enzyme quantity that oxidizes 1 mole of substrate per minute under the above reaction conditions.
3. Results and discussion 3.1. Proposal of mutation residue from structural studies and comparison with other FAODs Based on the previously proposed 3D structure model of N1-1 FAOD and enzyme–substrate docking models, several amino acid residues around the putative active site region, which are likely to play important roles in the enzymatic reaction and substrate recognition were identified [20]. His51 was identified as a residue involved in the recognition of the carboxyl groups of substrates [1,20]. Most of the other residues located around the putative active site of the structural model of N1-1 FAOD were relatively wellconserved in eukaryotic FAODs; however, His51 was not conserved (Fig. 1), and neither was Asn354 [1]. The eukaryotic FAODs can be classified into three groups based on their substrate specificities: FAODs showing a high preference for (i) ␣-glycated, (ii) -glycated amino acids, and (iii) FAODs able to oxidize ␣- and -glycated amino acids at comparable levels (Fig. 1). Interestingly, only the FAODs of the first group, i.e., ␣-fructosyl amino acid-specific FAODs, had a histidine residue at the position corresponding to the Asn354 of N1-1 FAOD, and this was consistent with the improvement of specificity
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S. Kim et al. / Enzyme and Microbial Technology 44 (2009) 52–56
Fig. 1. Alignment of the amino acid sequence around the His51 of N1-1 FAOD with other eukaryotic FAODs. The amino acid positions of the sequences are shown in parentheses. The enzymes are grouped according to substrate specificity: (i) preference for ␣-fructosyl amino acids, (ii) preference for -fructosyl amino acids, and (iii) similar activity with both ␣- and -glycated amino acids. The sequences are those of FAOD-P from Penicillum janthinelum AKU 3413, FAOD-U from Ulocladium sp. JS-103, FPOX-E from Eupenicillum terrenum ATCC 18547, FPOX-C from Coniochaeta sp. NISL 9330, FAOD-F from Fusarium oxysporum NBRC 9972, FAOD-A from Aspergillus terreus GP1, Amadoriases I from Aspergillus fumigatus, FAOD-Ao1 from Aspergillus oryzae, Amadoriases II from Aspergillus fumigatus, FAOD-Ao2 from Aspergillus oryzae and N1-1 FAOD from Pichia sp. N1-1. The arrowhead indicates the residues corresponding to the His51 of N1-1 FAOD. Identical amino acids are highlighted in black or dark gray and conserved amino acids in light gray.
for f-␣ Val observed in the Asn354His mutant FAOD which we created recently [1]. However, no histidine or positively charged amino acid was observed at the position corresponding to the His51 of N1-1 FAOD in any other FAODs, and there was also no particular amino acid conservation at this position among the FAODs of each group (Fig. 1). This led us to modify His51 in order to investigate the role of this residue on the enzymatic reaction, and it was expected that His51 mutants with various enzymatic properties would be obtained. 3.2. Site-directed mutagenesis of His51 We carried out the amino acid substitution of His51 with 19 other amino acids. As shown in Table 2, most of the created mutants showed no significant change in substrate specificity and decreased activity toward f-␣ Val, except for the substitutions with Lys, Arg, Trp and Tyr. His51Lys showed higher activity toward f-␣ Val compared to the wild-type and decreased activity toward f- Lys; i.e., it showed a slightly improved substrate specificity for f-␣ Val. Table 2 Activities and substrate specificities of crude wild-type and His51 mutant FAODs Activitya (mU mg−1 )
Wild-type His51Lys His51Arg His51Trp His51Tyr His51Ala His51Leu His51Ile His51Val His51Phe His51Cys His51Met His51Asn His51Gln His51Ser His51Thr His51Asp His51Glu His51Gly His51Pro
f-˛ Val
f-ε Lys
110 (100%) 170 (152%) 79 (72%) 37 (34%) 62 (56%) 7.2 (7%) 27 (24%) 17 (15%) 41 (38%) 50 (46%) 33 (30%) 23 (21%) 1.0 (1%) 10 (10%) 3.2 (3%) 14 (13%) 3.2 (3%) 1.1 (1%) 0.20 (0%) n.d.
340 (100%) 280 (80%) 24 (7%) 36 (10%) 61 (18%) 59 (17%) 110 (33%) 90 (26%) 140 (40%) 77 (22%) 200 (57%) 74 (22%) 89 (26%) 23 (7%) 64 (19%) 99 (29%) 4.5 (1%) 9.4 (3%) 9.1 (3%) n.d.
Activity for f-ε Lys/activity for f-˛ Val
3.2 1.5 0.31 0.96 0.99 8.2 4.2 5.4 3.4 1.5 5.9 3.3 88 2.2 20 7.2 1.4 8.3 45 –
The activities which could not be detected are represented as “n.d.”. a The activity was determined with 1 mM f-˛ Val or f-ε Lys in 5 mM PPB.
His51Arg showed improved substrate specificity for f-␣ Val (activity for f- Lys/activity for f-␣ Val: 0.31) as compared with the wild-type (3.2), maintaining relatively high activity toward f-␣ Val. His51Trp and His51Tyr both showed improved substrate specificity for f-␣ Val (0.96 for His51Trp and 0.99 for His51Tyr); however, their activities toward f-␣ Val markedly decreased. His51 was predicted to recognize the carboxyl group of substrates in the N1-1 FAOD substrate-binding model [20]. It was expected that several mutant FAODs with various properties would be obtained by introducing mutations at His51, according to the nature of the new amino acid. Substitutions with Gly, Ser, or Glu resulted in markedly decreased activities toward both substrates, although other eukaryotic FAODs have theses amino acids at the position corresponding to His51 of N1-1 FAOD. No significant amino acid sequence similarities in this region (His51 to Arg66 in N1-1 FAOD, Fig. 1) suggests that the role of His51 in N1-1 FAOD may be unique. Since the substitution with acidic amino acids (Asp and Glu) or a small amino acid (Gly) resulted in less than 5% of the wild-type activity toward both substrates, it is supposed that the interaction between His51 and the carboxyl groups in substrates is crucial for the activity of this FAOD. On the other hand, mutants, which were created by substituting His51 with amino acids having a basic side chain, His51Lys and His51Arg, retained the high activity toward f-␣ Val. This result suggests that electrostatic interaction plays a crucial role in the recognition of f-␣ Val. Mutations with hydrophobic amino acids showed activities (>15%) toward both substrates. It was considered that the hydrophobicity of the active site may also be important for the recognition of the substrates. However, alteration to bulky residues (Arg, Trp, Phe) resulted in markedly decreased activities toward f- Lys compared with f-␣ Val. According to our enzyme–substrate docking model, the predicted distance between His51 and the carboxyl group of f-␣ Val is longer than that between His51 and the carboxyl group of f- Lys [1]. Steric hindrance between f- Lys and the bulky side chains may be elicited by the mutations, and it was suggested that appropriate bulkiness of the amino acids in this position is important for the interaction with the substrate and may affect the substrate specificity. Since His51Lys and His51Arg showed improved activity and substrate specificity toward f-␣ Val, respectively, we further purified and characterized these two His51 mutant FAODs. As shown in Table 3, His51Lys showed an increased Vmax Km −1 value for f-˛ Val, 4.2 U mg−1 mM−1 , compared to the wild-type (1.7 U mg−1 mM−1 ). The Vmax Km −1 value for f-ε Lys of His51Lys,
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Table 3 Kinetic parameters of purified wild-type and mutant FAODs f-˛ Val
Wild-type His51Lys His51Arg His51Lys/Asn354His His51Arg/Asn354His Asn354His [1] Asn354Lys [1] a
f-ε Lys
Km (mM)
Vmax (U mg
3.9 2.4 11 0.4 2.4 0.6 2.0
6.5 10 17 11 15 16 25
−1
)
Vmax Km
−1
−1
(A) (U mg
−1
mM
1.7 4.2 1.5 28 6.3 27 13
)
B/A
Km (mM)
Vmax (U mg
0.9 2.0 18 27 –a 3.9 15
9.5 15 8.5 33 –a 16 21
−1
)
Vmax Km 11 7.5 0.47 1.2 –a 4.1 1.4
−1
−1
(B) (U mg
−1
mM
) 6.5 1.8 0.31 0.043 –a 0.15 0.11
Not determined.
7.5 U mg−1 mM−1 , was slightly lower than the 11 U mg−1 mM−1 of the wild-type; this means that the substrate specificity (Vmax Km −1 for f- Lys/Vmax Km −1 for f-˛ Val) was improved about 3.6-fold (1.8 for His51Lys, 6.5 for the wild-type). His51Arg retained the Vmax Km −1 value for f-˛ Val, 1.5 U mg−1 mM−1 , and had a greatly decreased Vmax Km −1 value for f-ε Lys, 0.5 U mg−1 mM−1 , compared to the wild-type; this indicates that the substrate specificity was drastically improved, by about 21 times (0.31 for His51Arg). Lys and Arg are basic amino acids with relatively bulky side chains. The positive charge and the appropriate bulkiness of the side chain of Lys lent His51Lys a considerably enhanced activity toward f-˛ Val and a slightly decreased activity toward f-ε Lys. In the case of His51Arg, steric hindrance may have played a particularly strong role in the interaction between f-ε Lys and Arg; this resulted in a remarkable decrease in the mutant enzyme’s activity toward f-ε Lys. 3.3. Characterization of His51Lys/Asn354His and His51Arg/Asn354His We then combined His51Lys and His51Arg with the previously reported Asn354His mutation [1] to improve the substrate specificity. As shown in Table 3, His51Lys/Asn354His showed a greatly increased Vmax Km −1 value for f-˛ Val, 28 U mg−1 mM−1 , and a greatly decreased Vmax Km −1 value for f-ε Lys, 1.2 U mg−1 mM−1 ; this means that the substrate specificity was improved about 125fold (0.043). His51Arg/Asn354His showed a 3.7-fold increase in the Vmax Km −1 value for f-˛ Val, 6.3 U mg−1 mM−1 , compared with the wild-type, and had almost no activity toward f-ε Lys. By combining the two amino acid substitutions, His51Arg and Asn354His, we succeeded in constructing an engineered FAOD, which has the cumulative effect of each single mutation. These two double-mutant FAODs showed a greater improvement in specificity for f-˛ Val over f-ε Lys than the wild-type and the single mutants even at low (5 mM) PPB concentration. The resulting double mutants are expected to be applied to the practical enzymatic measurement of HbA1c . Recently, the improvement of the substrate specificity of FAOD from Ulocladium sp. (FAOD-U) and Fusarium oxysporum (FAOD-F) has been reported [22,23]. However, these achievements depended on the random mutagenesis approach, and the substrate specificities toward their original preferred substrates (f-␣ Val for wild-type FAOD-U, f- Lys for wild-type FAOD-F) were improved. Furthermore, the discussion about the effect of the resulting mutation was not sufficient, because there was no information on the putative active site or the 3D structure. In this study, the constructed double mutants showed high specificity toward f-␣ Val, while wildtype N1-1 FAOD showed higher activity toward f- Lys than f-␣ Val. Furthermore, it is considered that our achievement is a result of a rational design approach based on the information of the proposed original structural model.
The mutant FAODs His51Lys/Asn354His and His51Arg/ Asn354His showed improved substrate specificity toward f␣ Val, which means that they have a great potential for use in the measurement of HbA1c in whole blood, because the interference from glycated albumin would be greatly reduced. In addition to creating mutant FAODs that are expected to be applied in the measurement of HbA1c , we have succeeded in combining two mutations in order to produce double mutants with a cumulative effect of the mutations. We demonstrated that our original structural model was useful for the engineering of N1-1 FAOD, and it is expected that various mutant FAODs with improved properties, such as peptidase resistance and activity, will be created based on this information. Furthermore, various mutations can be combined to create an ideal FAOD, and this approach will contribute to the development of enzyme-based sensing systems for glycated proteins. References [1] Miura S, Ferri S, Tsugawa W, Kim S, Sode K. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Eng Des Sel 2008;21:233–9. [2] Klenk DC, Hermanson GT, Krohn RI, Fujimoto EK, Mallia AK, Smith PK, et al. Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences. Clin Chem 1982;28:2088–94. [3] Johnson RN, Metcalf PA, Baker JR. Fructosamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin Chim Acta 1983;127:87–95. [4] Iberg N, Flückiger R. Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites. J Biol Chem 1986;261:13542–5. [5] Yoshida N, Sakai Y, Serata M, Tani Y, Kato N. Distribution and properties of fructosyl amino acid oxidase in fungi. Appl Environ Microbiol 1995;61: 4487–9. [6] Yoshida N, Sakai Y, Isogai A, Fukuya H, Yagi M, Tani Y, et al. Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins. Eur J Biochem 1996;242:499–505. [7] Takahashi M, Pischetsrieder M, Monnier VM. Isolation, purification, and characterization of amadoriase isoenzymes (fructosyl amine-oxygen oxidoreductase (EC 1.5.3) from Aspergillus sp. J Biol Chem 1997;272:3437–43. [8] Takahashi M, Pischetsrieder M, Monnier VM. Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine-oxygen oxidoreductase, (EC1.5.3) from Aspergillus fumigates. J Biol Chem 1997;272:12505–7. [9] Hirokawa K, Gomi K, Bakke M, Kajiyama N. Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi. Arch Microbiol 2003;180: 227–31. [10] Akazawa S, Karino T, Yoshida N, Katsuragi T, Tani Y. Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae. Appl Environ Microbiol 2004;70:5882–90. [11] Sode K, Ishimura F, Tsugawa W. Screening and characterization of fructosyl– valine-utilizing marine microorganisms. Mar Biotechnol 2001;3:126–32. [12] Horiuchi T, Kurokawa T, Saito N. Purification and properties of fructosyl amino acid oxidase from Corynebacterium sp. 2-4-1. Agric Biol Chem 1989;53:103–10. [13] Ferri S, Sakaguchi A, Goto H, Tsugawa W, Sode K. Isolation and characterization of fructosyl-amine oxidase from an Arthrobacter sp. Biotechnol Lett 2005;27:27–32. [14] Ferri S, Miura S, Sakaguchi A, Ishimura F, Tsugawa W, Sode K. Cloning and expression of fructosyl-amine oxidase from marine yeast Pichia sp. N1-1. Mar Biotechnol 2004;6:625–32. [15] Tsugawa W, Ishimura F, Ogawa K, Sode K. Development of an enzyme sensor utilizing a novel fructosyl amine oxidase from a marine yeast. Electrochemistry 2000;68:869–71.
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