1 Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 21, Suita, Osaka 565-0871 and 4 Department of Biological Science and Technology, Tokai University, Nishino, Numazu, Shizuoka 410-0321, Japan
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Abstract |
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Keywords: cholesterol oxidase/site-directed mutagenesis/Streptomyces/structural characterization/substrate specificity
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Introduction |
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The primary sequence of ChoAS was aligned with that of ChoAB and has served along with the three-dimensional structure of the cholesterol oxidase catalytic domain, as an important guide for various protein engineering studies of cholesterol oxidase. Thermostable cholesterol oxidase with a broad optimum pH range has a potential for clinical applications owing to its advantage of having a faster reaction rate. Therefore, we previously investigated the molecular basis of the difference in heat stability between the structurally similar ChoAS and ChoAB. Genetic engineering by random mutagenesis improved the thermostability of cholesterol oxidase activity (Nishiya et al., 1997). The three-dimensional structure can serve as a basis for analyzing the relationships between structure and function for more thermostable or high-affinity mutant forms of ChoAS that can be generated by site-directed and random mutagenesis (Nishiya et al., 1997
; Yamashita et al., 1998
; Nishiya and Hirayama, 1999
). The aim was not only to generate a more thermostable ChoAS that could have commercial potential, but also to gain some insight into the structurefunction relationships. To assess the role of specific amino acid residues in the thermostability of the enzyme based on the results described previously (Nishiya et al., 1997
), we generated several enzymes with mutated amino acids near the FAD- or substrate-binding site in the structure of ChoAS that were not identical with amino acids in ChoAB. The characterization of these mutant enzymes revealed that the V145 residue is an important determinant of thermostability (Murooka et al., 1998
).
In this study, we introduced mutations in cholesterol oxidase to determine whether its activity can be altered by genetic engineering. All mutations were characterized by their substrate specificities and kinetic parameters. Some of the mutant enzymes had reduced catalytic activity and one had a high catalytic activity for cholesterol and pregnenolone. To our knowledge, this is the first report of a change in the substrate specificity of cholesterol oxidase by site-directed mutagenesis.
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Materials and methods |
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The following steroids were used as substrates: cholesterol (Nakalai Tesque, Kyoto, Japan), pregnenolone, dihydrocholesterol, dehydroisoandrosterone, estrone and 5-cholesten-3ß -ol-7-one (Steraloids, Wilton, NH). Enzymes for the manipulation of DNA were purchased from Toyobo (Osaka, Japan) or Takara Shuzo (Kusatsu, Shiga, Japan) and were used according to the manufacturer's instructions.
Strains, plasmid, phage and culture conditions
E.coli strains BMH71-18 and MV1184 were used for site-directed mutagenesis. E.coli JM109 was used as the host for manipulations of recombinant DNA, for the production of wild-type and mutant cholesterol oxidases and for site-directed mutagenesis. Plasmid pCO117 (Nomura et al., 1995), containing the choA gene, and a phage vector that corresponded to the replicative form DNA of M13tv19 (Takara Shuzo) were used for the construction of additional plasmids and site-directed mutagenesis, respectively. Recombinant strains of E.coli were grown in the LB medium (Sambrook and Russel, 2001
) supplemented with ampicillin (100 µg/ml).
Manipulation and analysis of DNA
Purification of DNA, digestion with restriction enzyme, ligation, agarose gel electrophoresis and transfomation of competent E.coli cells were performed according to the method of molecular cloning described previously (Sambrook and Russel, 2001). Nucleotide sequences were determined with an ALF DNA sequencer (Amersham Pharmacia Biotech UK, Amersham, Bucks., UK).
Site-directed mutagenesis and construction of expression plasmids
The choA gene of Streptomyces sp. was subcloned from pCO117 into bacteriophage M13tv19. Site-directed mutagenesis was performed with a MutanG kit (Takara Shuzo), designed with reference to the procedure of Kramer et al. (1984), according to the manufacturer's instructions. The presence of desired mutations and the absence of adventitious base changes were verified by DNA sequencing. The following oligonucleotides were used for mutagenesis: 5'-GCT CGG CAG CTT CCC ATG GCT CGA CGT CGT-3' (L117P); 5'-TTC CTC TGG GCC GAC GTC GTT AAC CGG AAC-3' (L119A); 5'-AGC TTC CTC TGG TTC GAC GTC GTCA-3' (L119F); 5'-CCA GCT GTC GGT ATA CCA GGG CCG CGG CGT-3' (V145Q); 5'-CCA GAT GTC GGT ATA CGA TGG CCG CGG CGT-3' (V145D); 5'-CCA GAT GTC GGT ATA CGA GGG CCG CGG CGT-3' (V145E); 5'-CGG ATC GTC TTG ACA CGG TGC AGG GTC TGG-3' (Q286R); 5'-GCG CGG GCT GGG GCA ACA ACG GCA ACA TC-3' (P357N); 5'-GCG CCC ACC AGT CGA CCA TCC CCG CCC TCG-3' (S379T); 5'-GCG CCC ACC AGT CGA CCA GCC CCG CCC TCG-3' (S379A); 5'-GCG CCC ACC AGT CGA CCA GTC CCG CCC TCG-3' (S379V); 5'-GCG CCC ACC AGT CGA CCA CGC CCG CCC TCG-3' (S379R); and 5'-GCG CCC ACC AGT CGA CCA GAC CCG CCC TCG-3' (S379D). Mutated DNA was isolated as the double-stranded replicative form from E.coli MV1184, which was infected by M13tv19 and digested with appropriate restriction enzymes. Each fragment of mutated DNA was reintroduced into plasmid pCO117.
Expression and purification of recombinant cholesterol oxidases
For the preparation of ChoAS, E.coli JM109 cells carrying an expression plasmid that included a wild-type or a mutant choA were cultured in the LB medium supplemented with ampicillin (100 µg/ml) and isopropyl-ß-D-thiogalactopyranoside (IPTG; final concentration, 0.1 mM) at 37°C for 15 h. Recombinant wild-type and mutant enzymes were purified by a previously described procedure (Nomura et al., 1995) with the following modifications. Harvested cells were washed with 100 mM phosphate buffer (pH 7.0), suspended in the same buffer and then disrupted by sonication. After centrifugation of the sonicate, the supernatant was used as crude enzyme for measurements of cholesterol oxidase activity. For further purification, we used cholesterol affinity chromatography (Yamashita et al., 1998
) and hydrophobic column chromatography on butyl-Toyopearl 650M (Tosoh, Tokyo, Japan) to purify the wild-type and mutant enzymes to homogeneity. Concentrations of protein were calculated from the absorbance of samples at 280 nm; more precise determinations of the protein concentration were made as described previously (Lowry et al., 1951
), using bovine serum albumin as the standard. The purity of each preparation of enzyme was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) on a gel that contained 10% (w/v) acrylamide, as described previously (Laemmli, 1970
), with subsequent staining with Coomassie Brilliant Blue R-250. Five proteins were used as molecular mass standards: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and soybean trypsin inhibitor (20.1 kDa) (LMW marker; Amersham Pharmacia Biotech UK).
Assays of enzymatic activity
Enzymatic activity was examined as described previously (Allain et al., 1974). Solutions of enzyme (0.51.0 U/ml) were prepared by dilution of extracts with 10 mM phosphate buffer (pH 7.0). For determination of cholesterol oxidase activity, the appearance of quinonimine dye, which is formed by the reaction with 4-aminoantipyrine, phenol and peroxidase, was measured spectrophotometrically at 500 nm. One unit of activity was defined as the activity associated with the formation of 1 µmol of hydrogen peroxide (0.5 µmol of quinonimine dye) per minute at 37°C and pH 7.0.
Kinetic studies
When an enzyme-catalyzed reaction can be represented by the integrated MichaelisMenten equation, the maximum velocity and Michalis constant can be determined from a single experiment. This type of data processing is possible for stable enzymes that catalyze an irreversible reaction with a single substrate and in which none of the reaction products is inhibitory. We used reaction mixtures that contained 670, 335, 168, 84, 42 or 21 mM substrate to calculate values of kcat and Km and analyzed the kinetic data by constructing double-reciprocal plots.
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Results |
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The amino acid sequence identity between the enzyme from Streptomyces (ChoAS) and that from Brevibacterium (ChoAB) is 59% (Figure 2). Major residues, which are located in the active site, are completely conserved between the two enzymes. Thus, the structure of ChoAS is essentially identical with that of ChoAB. Moreover, the hydrogen-bonding network formed around H2O541 of the two native structures is superimposable. It seems reasonable to assume that the difference in activities between the two enzymes is not due to differences in the catalytic groups in the active site. To determine amino acid residues that are key to the substrate specificity of ChoAS, we selected six amino acid residues, L117, L119, V145, Q286, P357 and S379, which are located within 4.5 Å of the FAD- or substrate-binding sites of ChoAS (Figure 3
). Many amino acid residues around the FAD- and substrate-binding sites are strongly conserved in ChoAS and ChoAB, whereas the selected amino acid residues of ChoAS are different from those of ChoAB. Since the kcat/Km values of ChoAS, 1.5x107 and 2.5x104 M-1 s-1 for cholesterol and dehydroisoandrosterone (AND), respectively, are different from those of ChoAB, 5.0x105 and 1.2x104 M-1 s-1, respectively (Yue et al., 1999
), amino acid residues in ChoAS were substituted for those in ChoAB as a basic strategy. Moreover, L117 and L119 in the loop region, which seems to act as a lid over the active site, were replaced with Pro for less flexibility of the loop region and with Ala for construction of a small cavity between the loop region and the substrate pocket, respectively. Mutations causing substitutions by L117P, L119A, L119F, V145Q, Q286R, P357N and S379T were introduced to the choA gene using site-directed mutagenesis and confirmed by their respective DNA sequencing. These substitutions were based on the amino acid sequence alignment and comparisons with that of ChoAB. We analyzed the productivities of these mutant enzymes and their activities with cholesterol as the substrate in a crude extract of transformed E.coli cells that carried the plasmid that included each respective gene. The specific activities of mutant L119A, L119F, V145Q, Q286R, P357N and S379T enzymes were similar to that of the wild-type enzyme (0.39 U/mg protein). However, the mutant L117P enzymes had no activity, even when concentrated 100-fold (data not shown). Therefore, no further characterization of this L117P mutant was performed.
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The wild-type and L119A, L119F, V145Q, Q286R, P357N and S379T mutant enzymes had cholesterol oxidase activity and could therefore be purified by cholesterol affinity chromatography and hydrophobic column chromatography on butyl-Toyopearl 650M. The amounts of six mutant enzymes, and also the recombinant wild-type ChoAS, were estimated based on results of SDSPAGE analysis. Each preparation of purified enzyme yielded a single band with the same mobility as that of the wild-type enzyme (Figure 4). The molecular mass of each enzyme was determined from its mobility and was found to be 55 kDa, confirming the result of a previous report (Nomura et al., 1995
).
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We examined the substrate specificities of the wild-type and mutant enzymes for several sterols, such as cholesterol, pregnenolone, dihydrocholesterol, AND, estrone and 5-cholesten-3ß-ol-7-one (Table I). The wild-type enzyme and L119A, L119F, Q286R and P357N mutant enzymes had comparatively high activity for cholesterol, moderate activity for pregnenolone and dihydrocholesterol and little or no activity for AND, estrone and 5-cholesten-3ß-ol-7-one. The rank order of activities of these mutant enzymes for the various substrates was almost the same as that of the wild-type enzyme. However, the mutant V145Q enzyme showed low activity for all of substrates examined, whereas the activity of the mutant S379T enzyme for pregnenolone was 1.8-fold higher than that for cholesterol. These results indicate that the V145 and S379 residues might be important for catalytic activity including specificites.
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The kinetic parameters for the recombinant wild-type and mutant ChoAS were determined at 37°C using 21670 µM cholesterol and pregnenolone as substrates. The kcat and Km values for recombinant wild-type ChoAS were 40 s-1 and 2x10-2 µM, respectively; the kcat value was similar to and the Km was 150-fold lower than those obtained for the recombinant wild-type ChoAS reported previously (Yue et al., 1999). The difference in Km value between the present and previous data seems to be due to the different surfactants used to dissolve the sterols, Triton X-100, isopropyl alcohol and polyoxyethlene 9 lauryl ether (Nishiya et al., 1997
; Sampson et al., 1998
). We compared the kcat and Km values of all of the purified mutant enzymes, with cholesterol and pregnenolone as substrates, using those of the wild-type enzyme (Table II
). The catalytic efficiency (kcat/Km) of the mutant L119A, L119F, V145Q, Q286R and P357N enzymes with cholesterol and pregnenolone was lower than that of the wild-type enzyme, independent of their kcat and Km values. In contrast, the kcat/Km of the mutant S379T enzyme for cholesterol and pregnenolone was 1.8- and 6.0-fold higher, respectively, than that of the wild-type enzyme (Table II
).
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Discussion |
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The L117P substitution might have caused the disruption of the loop structure and the resultant mutant enzyme seems to have lost its activity for cholesterol. We examined the synthesis of mutant L117P cholesterol oxidase by western blotting. The production yield of mutant L117P cholesterol oxidase was almost the same as that of the wild-type enzyme (data not shown). Mutant L119A and L119F enzymes showed almost the same substrate specificity and kinetic parameters as those of the wild-type enzyme. However, both mutant enzymes were more thermally unstable than the wild-type enzyme (Murooka et al., 1998). Taken together, the loop region appears to be more important for thermostability than for substrate specificity.
We used random and site-directed mutagenesis in an attempt to engineer ChoAS to improve the thermostability based on the structure homology model (Fujii et al., 1994). Both methods produced mutant enzymes, V145E and V145D, with improved thermostability compared with the wild-type and the V145Q mutant enzyme; moreover, the combination of multiple mutations gave better results (Nishiya et al., 1997
; Murooka et al., 1998
). Recently, the double-mutant V145E/G405S enzyme was reported to have a high Km value for cholesterol. However, the effects of the site of mutation on the low affinity for the substrate were not examined in detail (Nishiya and Hirayama, 1999
). In this study, we found that the Km value of the mutant V145E enzyme for cholesterol was 34-fold higher than that of the wild-type enzyme. These results suggest that the high Km value of the double-mutant V145E/G405S enzyme might have resulted from the V145E mutation. The V145 residue is located near the FAD-binding site and not near the substrate-binding site (Figure 2
). Since the interaction of D145 or E145 with D134 and R147 might be more stable than that of V145 or Q145, the V145D and V145E mutant enzymes seem to restrict the insertion of cholesterol into the substrate-binding pocket to a greater degree than that of the wild-type and the V145Q mutant enzyme (Nishiya et al., 1997
). These mutational analyses suggest that a more thermostable mutant of ChoAS shows a much lower catalytic efficiency compared with that of the wild-type enzyme.
The three-dimensional structures of ChoAS and ChoAB reveal that amino acid residues around the substrate (AND) are almost the same. However, the major difference between the two structures is that the cavity around the side chain of S379 in ChoAS is larger than that of T387 in ChoAB (Figure 5). This difference appears to translate into differences in kinetic parameters in the mutant S379T enzyme. The side chain of T379 in the S379T mutant enzyme presumably fits the cholesterol and pregnenolone molecules compared with that of S379 in ChoAS. Since a slightly larger size of the side chain of Val than that of Thr might have caused steric hindrance with the atoms of neighboring residues and substrate, the Km values of the S379V mutant enzyme for both cholesterol and pregnenolone were the same as those of the wild-type enzyme and larger than those of the S379T mutant enzyme (Table II
). The absence of bound cholesterol and any activities following S379D and S379R substitutions may be due to the hydrophilic and too large side chains of the amino acid residue 379 in the S379D and S379R mutant enzymes, disrupting spatial formation of an appropriate cavity around the Cß atom of 379 amino acid residues (data not shown). These results and the Km value of the S379A mutant enzyme for both substrates could be explained by the finding that the kcat value of the S379T mutant enzyme for pregnenolone appears to be slightly larger than that of the wild-type enzyme and the size of side chain in the amino acid residue 379 contributes to the improvement of the Km value of the S379T mutant enzyme for pregnenolone.
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Notes |
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2 To whom correspondence should be addressed. E-mail: yamashita{at}bio.eng.osaka-u.ac.jp
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Acknowledgments |
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References |
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Received September 25, 2001; revised February 19, 2002; accepted February 20, 2002.