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A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês

10.1074/jbc.m610320200

1083-351X

Akio Suemori, Masahiro Iwakura,

Enzyme Structure and Function

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties. We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties. As recently reported, our evolutional design method, namely, "quasi-additive adaptive walking with mutant data base" (QAW), 2The abbreviations used are: QAW method, quasi-additive adaptive walking method with mutant database; PHBH, p-hydroxybenzoate hydroxylase. provides a systematic and comprehensive combinatorial scheme for obtaining an improved protein with a single desired property (1Iwakura M. Maki K. Takahashi H. Takenawa T. Yokota A. Katayanagi K. Kamiyama T. Gekko K. J. Biol. Chem. 2006; 281: 13234-13246Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Because the previous work focused on a single property (enzyme activity), whether or not our QAW method could be extended to improve simultaneously multiple properties of any enzyme should be examined for generalization and availability as to industrial uses of our method. In a test study, we used p-hydroxybenzoate hydroxylase (PHBH) as a target enzyme and all Cys and Met residues as mutation sites, similar to the previous study (1Iwakura M. Maki K. Takahashi H. Takenawa T. Yokota A. Katayanagi K. Kamiyama T. Gekko K. J. Biol. Chem. 2006; 281: 13234-13246Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Among the 20 naturally occurring amino acids, only Cys and Met residues contain a sulfur atom in their side chains. Because of the high reactivity of sulfur in sulfhydryl and thiomethyl groups, proteins are easily oxidized and show molecular heterogeneity because of the formation of inter- and intra-molecular disulfide linkages, and changes in isoelectric points caused by the formation of methionine sulfoxide. Therefore, sulfur-free or reduced proteins should be resistant to oxidative damage and could be used in highly oxidizing environments such as those found in pollution treatment facilities. PHBH, which is a FAD-containing monooxygenase, catalyzes the hydroxylation of p-hydroxybenzoate to protocatechuate (main activity) (Fig. 1A) (2Entsch B. van Berkel W.J. FASEB J. 1995; 9: 476-483Crossref PubMed Scopus (183) Google Scholar, 3Palfey B.A. Moran G.R. Entsch B. Ballou D.P. Massey V. Biochemistry. 1999; 38: 1153-1158Crossref PubMed Scopus (86) Google Scholar) and plays an important role in degrading various aromatic compounds in the environment (4Harayama S. Kok M. Neidle E.L. Annu. Rev. 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Protein Sci. 2001; 10: 1712-1728Crossref PubMed Scopus (385) Google Scholar). PHBH has also been a subject of dissection and reconstruction for the development of general catalysts with oxygen and electronic transfer. From a clinical viewpoint, various assay methods for serum cholinesterase activity for the diagnosis of a liver function have been proposed. One popular method includes determination of p-hydroxybenzoate, the reaction product of cholinesterase, using PHBH (40Ashihara Y. Kasahara Y. Sugiyama M. Harada T. J. Biochem. (Tokyo). 1983; 94: 11-15Crossref PubMed Scopus (19) Google Scholar). However, the sub-activity of the enzyme reduces the accuracy of the method. Thus, raising of the reaction specificity by simultaneously raising the main activity and reducing the sub-activity are desired as well as protein stability, as usually desired for protein engineering. Here we report an attempt at simultaneous improvement of these properties of PHBH from Pseudomonas fluorescens NBRC 14160 by means of combinatorial searches starting from a Cys and Met single mutant pool. A number of improved mutants were effectively obtained after seven combinatorial searches, four with one selection parameter, two with two selection parameters, and one with four selection parameters. In addition, comparison of various combinatorial conditions was carried out. Materials—The PCR, DNA extraction, DNA purification, DNA sequencing, and nickel-nitrilotriacetic acid protein purification kits were purchased from Qiagen; the protein extraction kit was from Novagen; and S-Gal/LB Agar Blend was purchased from Sigma. Sodium p-hydroxybenzoate was from Fluka, and other aromatic carboxylic acid compounds were from Wako Pure Chemical Industries. The following PCR primers were obtained from Jbios. Overexpression—To isolate the gene encoding PHBH (pobA), chromosomal DNA from P. fluorescens NBRC 14160 (DDBJ/DAD accession numberBAB20910) was used as the template (20Suemori A. Ozawa M. Iwakura M. Biotechnol. Lett. 2001; 23: 489-495Crossref Scopus (4) Google Scholar). To generate a 1297-bp fragment containing the respective pobA sequence with an EcoT22I-restriction site at the N terminus and a His6 tag and EcoRI restriction site at the C terminus, primers SD/EcoT22I/pobA-N and pobA/H6/EcoRI-C were used. A 1238-bp fragment was amplified by PCR with 35 cycles of melting at 94 °C for 60 s, annealing at 55 °C for 60 s, and comprising extension at 72 °C for 90 s. The amplified DNA fragment was purified and then used as the template for the following PCR. To obtain a 1278-bp fragment containing the P35 and SD regions upstream of pobA, primers P35/SD/EcoT22I-N and pobA/H6/EcoRI-C were used. PCR and purification of the resulting fragment were performed as described above. To generate a 1307-bp fragment containing a PstI restriction site upstream of the P35 region, PCR was carried out with the 1278-bp fragment as the template and primers PstI/P35-N and pobA/H6/EcoRI-C. The 1307-bp fragment was purified, digested with restriction enzymes (PstI and EcoRI), and then repurified. The digested fragment was ligated into similarly digested pUC19, transformed into Escherichia coli JM109 cells by means of heat shock, and then plated on LB agar plates containing ampicillin and p-hydroxybenzoate. Transformants exhibiting the formation of a violet color because of the production of protocatechuate were isolated as single colonies. DNA was isolated from the resulting transformants and sequenced using a Big Dye sequencing kit. The plasmid containing pobA with the three restriction sites (PstI, EcoT22I, and EcoRI), two regions (P35 and SD), and His6 tag was designated as pPHBH. Mutant Libraries—To create libraries of single mutants as to various Cys and Met positions, the pPHBH plasmid was used as a PCR template. To produce a fragment of C152X-A, two primers (PstI/P35-N and C152X-C) were used, and to produce a fragment of C152X-B, two other primers (C152X-N and pobA/H6/EcoRI-C) were used. Amplified DNA fragments C152X-A and C152X-B were purified and used as templates for a primerless PCR. After 10 cycles of the same condition, two primers (P35/SD/EcoT22I-N and pobA/H6/EcoRI-C) were added for an additional 35 cycles to enhance amplification under the complete 1238-bp fragment. The 1238-bp fragment containing C152X mutations was purified and digested with restriction enzymes (EcoT22I and EcoRI). For functional screening, the digested fragment was ligated into a vector (pPHBH digested at EcoT22I and EcoRI sites). Transformation, overexpression, plasmid extraction, and identification of C152X mutations were carried out as described above. To obtain libraries of double variants, for example, M98V/M110S, the M98V single mutant, as a template, two primers (P35/SD/EcoT22I-N and M110S-C) for M98V/M110S-A, and two primers (M110S-N and pobA/H6/EcoRI-C) for M98V/M110S-B, respectively, were used to generate M98V/M110S. Characterization of Enzymes—E. coli JM109 cells were transformed with the plasmid containing mutations in pobA, and a single colony was used to inoculate 200 ml of LB medium containing ampicillin. After overnight incubation, the cells were harvested by centrifugation, and the cell pellet was lysed with phosphate buffer. The cell-free supernatant was purified using His6 tag and a nickel-nitrilotriacetic acid protein purification kit (20Suemori A. Ozawa M. Iwakura M. Biotechnol. Lett. 2001; 23: 489-495Crossref Scopus (4) Google Scholar). PHBH activity was monitored by following the rate of p-hydroxybenzoate-dependent oxidation of NADPH at 340 nm at 25 °C using as the assay mixture the following: 100 mm HEPES/NaOH buffer (pH 8.0) containing 0.2 mm sodium p-hydroxybenzoate (or an other aromatic compound), 0.2 mm NADPH, 2 μm FAD, and a suitable amount of enzyme solution in a total volume of 2 ml (20Suemori A. Ozawa M. Iwakura M. Biotechnol. Lett. 2001; 23: 489-495Crossref Scopus (4) Google Scholar). Thermal stability was calculated by measuring the remaining activity after the purified enzyme in 100 mm HEPES/NaOH buffer (pH 8.0) was left standing at 50 °C for 30 min and then cooled. A Strategy for a Combinatorial Method—In an attempt at simultaneous improvement of enzymatic properties using the QAW concept, a comprehensive combination with a mutant data base (1Iwakura M. Maki K. Takahashi H. Takenawa T. Yokota A. Katayanagi K. Kamiyama T. Gekko K. J. Biol. Chem. 2006; 281: 13234-13246Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), we devised a strategy of combinatorial mutations starting from available single mutations. The combinatorial method for generating mutants used in this study does not use the combination of only the single best mutant at various positions. Rather, our method has the following key features: (i) mutants (if possible, three or more) are selected from a single and multiple mutant data base using various selection parameters; (ii) all combinations of the selected mutants are generated to construct a secondary mutant data base; and (iii) iterative selection, combination, and construction of subsequent mutant data bases are carried out until multiple mutants with the desired properties are obtained. Such combinatorial mutations as to various residues using multiple selection parameters can be referred to as multidimensional or matrix combination. It is also notable that the quadruple mutants were not obtained by chance from a random mutant library. It should be possible to reproducibly reconstruct any of the variants with improved properties, because all of the combinations were carried out based on the single and multiple mutant data bases. Construction of a Single Mutant Data Base—The 397-amino acid sequence of PHBH from P. fluorescens NBRC 14160 contains the following four Cys and eight Met residues in addition to a Met residue at the N terminus: Cys-152, Cys-158, Cys-211, and Cys-332 and Met-52, Met-65, Met-98, Met-110, Met-213, Met-276, Met-34, and Met-349 (Fig. 2) (20Suemori A. Ozawa M. Iwakura M. Biotechnol. Lett. 2001; 23: 489-495Crossref Scopus (4) Google Scholar). Substitution of each Cys and Met as target positions with one of the other 18 naturally occurring amino acids resulted in 216 active and stable single mutants. These single mutants were examined as to three properties as follows: hydroxylase activity toward p-hydroxybenzoate (main activity; units/mg), protocatechuate-dependent NADPH oxidase activity (sub-activity; units/mg), and thermal stability (%). Subsequently, the ratio of sub-activity to main activity (reaction specificity; %) was calculated based on the main activity and sub-activity. Using these four properties, a data base of single mutants as to Cys and Met sites was constructed (supplemental Table 1). Fig. 3 shows the distribution of the 216 single mutants with each of the four properties. As shown in Fig. 3 (A-D), there was a tendency that the substitution of Cys and Met residues with aliphatic amino acids caused the main activity and sub-activity values of the single variants to increase; however, their replacement with charged ones caused them to decrease. As shown in Fig. 3 (E-H), the mean and standard variation (shown in parentheses) values for the main activity, sub-activity, reaction specificity, and thermal stability for the 216 single mutants were similar to the wild type values: 14.7 (2.3) units/mg, 0.734 (0.126) units/mg, 5.0% (0.7%), and 31% (7%), respectively. In particular, only 28% of the single mutants exhibited reaction specificity values lower than that of the wild type, indicating that the distortion values of distribution for reaction specificity were rather high. A detailed study of the single mutants revealed a loose relationship between the main activity and sub-activity with a correlation coefficient of 0.648 (see supplemental Fig. 7 (A1)). As compared with the wild type, 50% of the 216 single mutants exhibited both lower main activity and lower sub-activity, 22% both higher main activity and higher sub-activity, 23% lower main activity and higher sub-activity, and only 5% higher main activity and lower sub-activity. This tendency suggested the difficulty of simultaneous alterations with increasing main activity and decreasing sub-activity. Combination Using One Selection Parameter—Combination of single mutants was carried out using only one selection parameter, main activity. First, eight single mutants with the highest main activity (M98V, M110S, M346G, M346A, M346I, M346E, M349A, and M349P) were selected from among the single mutants (Fig. 4a). All possible combinations of the selected eight single mutants were performed, resulting in 11 active and stable double variants (Fig. 4b). Next, four double variants with the highest main activity were selected and comprehensively combined. This combination provided three active and stable triple variants (Fig. 4c). Because all of the substituted positions in the triple variants (M98V/M110S/M346G, M98V/M10S/M346E, and M98V/M110S/M346A) were the same, no further combination was possible. The maximal main activity values for the selected single mutants and double and triple variants were 21.3 units/mg (M346G), 21.5 units/mg (M110S/M346G), and 22.6 units/mg (M98V/M110S/M346G), respectively. In addition, all of the triple variants exhibited not only higher main activity but also higher thermal stability than the wild type (Fig. 4c). On the other hand, the reaction specificity values of the multiple mutants were not significantly different from that of the wild type. A combinatorial experiment using only one selection parameter (sub-activity, reaction specificity, or thermal stability) was performed (data not shown). The experimental results indicated the same tendency, i.e. only the property used as the selection parameter was improved. Combination Using Two Selection Parameters—From the results of combination using one selection parameter, it was hypothesized that the combination of mutations causing both higher main activity and higher thermal stability should allow simultaneous and efficient improvement of two properties (main activity and thermal stability). Based on such a hypothesis, first, 27 single mutants were selected that showed both higher main activity and higher thermal stability as compared with the wild type. Next, from among the selected 27 single mutants, 4 single mutants with the highest main activity (M110S, M346G, M346E, and M349A) and 4 single mutants with the highest thermal stability (M52V, M98L, M213G and M276Q) were selected, respectively (Fig. 4d). Comprehensive combination of these selected 8 single mutations generated 15 active and stable double variants (Fig. 4e). From these double variants, the two with the highest main activity and the two with the highest thermal stability were selected, respectively. The selected four double variants were comprehensively combined, resulting in five active and stable triple and two quadruple variants (Fig. 4, f and g). Because all replacement positions in the two quadruple variants (M98L/M110S/M276Q/M346G and M98L/M110S/M276Q/M346E) were the same, no further combinations were possible. The maximal main activity and thermal stability values for the selected single mutants and the double, triple, and quadruple variants were 21.3 units/mg and 43% (M346G), 21.5 units/mg and 47% (M110S/M346G), and 23.5 units/mg and 51% (M98L/M110S/M276Q/M346G), respectively. In contrast, neither sub-activity nor reaction specificity was reduced, implying that only the two properties used as selection parameters were improved. Alternatively, to improve the reaction specificity through simultaneous enhancement of main activity and sub-activity, the combination by using two selection parameters (main activity and sub-activity) was performed. Twelve single mutants with both higher main activity and lower sub-activity than the wild type were selected (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M110P, M213G, M213L, M276Q, and M349A) (Fig. 4h). The selected 12 single mutants were comprehensively combined, which generated 15 active and stable double variants showing both higher main activity and lower sub-activity than the wild type (Fig. 4i). From among the 15 double variants, the 3 with the highest main activity and the 3 with the lowest sub-activity were selected, respectively. These six double variants were comprehensively combined, which produced one active and stable triple and eight quadruple variants (Fig. 4 (j and k)). Further combination of the quadruple variants did not yield active and stable quintuple variants. The maximal and minimal values of the four properties for the selected single mutants and double and quadruple variants were as follows: maximal main activity, 17.9 units/mg (M110I), 19.5 units/mg (M52Q/C211I and M52V/C211I), and 19.3 units/mg (M52Q/M110I/C152V/C211I); minimal sub-activity, 0.592 units/mg (M52V), 0.420 units/mg (M110I/M276Q), and 0.357 units/mg (M52Q/M110L/C211I/M276Q); and minimal reaction specificity, 3.3% (M52V), 2.4% (M110L/M276Q), and 2.1% (M52Q/C152V/C211I/M213G and M52Q/M110I/C152V/C211I). These results obviously indicated that both the main activity and sub-activity of multiple variants were simultaneously improved, and thus the reaction specificity was efficiently reduced. Combination Using All Four Selection Parameters—The two results of combinatorial experiments using two selection parameters led the combination by using all four selection parameters (main activity, sub-activity, reaction specificity, and therma