Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Mitsutoshi Toyama1, Mitsuo Yamashita1,2, Morihide Yoneda1, Andrzej Zaborowski1,3, Masaki Nagato1, Hisayo Ono1, Noriaki Hirayama4 and Yoshikatsu Murooka1

1 Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2–1, Suita, Osaka 565-0871 and 4 Department of Biological Science and Technology, Tokai University, Nishino, Numazu, Shizuoka 410-0321, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and Brevibacterium. Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (kcat/Km) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high kcat value and a low Km value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.

Keywords: cholesterol oxidase/site-directed mutagenesis/Streptomyces/structural characterization/substrate specificity


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cholesterol oxidase (3ß-hydroxysteroid oxidase; EC1.1.3.6) is a flavin adenine dinucleotide (FAD)-dependent bifunctional enzyme that catalyzes both the oxidation of cholesterol (5-cholesten-3ß-ol) to the temporary intermediate 5-cholesten-3-one, with the reduction of molecular oxygen to hydrogen peroxide, and the isomerization of the steroid with a trans A:B ring junction to produce 4-cholesten-3-one (Stadtman et al., 1954Go) (Figure 1Go). This enzyme is industrially important and useful for the clinical determination of serum cholesterol levels in combination with related enzymes (Allain et al., 1974Go). The enzyme is also used in the microanalysis of steroids in food samples and for distinguishing the steric configurations of 3-ketosteroids from the corresponding 3ß -hydroxysteroids.



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Fig. 1. Enzymatic reaction catalyzed by cholesterol oxidase with cholesterol, pregnenolone and dehydroisoandrosterone (AND) as the steroid substrates.

 
We previously cloned and sequenced the gene encoding cholesterol oxidase (choA) from Streptomyces sp. SA-COO (Murooka et al., 1986Go; Ishizaki et al., 1989Go) and the overproduction and secretion of Streptomyces cholesterol oxidase (ChoAS) in a Streptomyces host–vector system have been demonstrated (Molnár et al., 1993Go). The expression of the choA gene in Escherichia coli after genetic modification of E.coli cells has also been reported (Nomura et al., 1995Go). The gene encoding cholesterol oxidase (ChoAB) from Brevibacterium sterolicum was also cloned, sequenced and expressed in E.coli (Ohta et al., 1991Go, 1992Go). Moreover, the crystal structure of ChoAB, with and without the steroid substrate dehydroisoandrosterone (AND) at 1.8 Å resolution (Vrielink et al., 1991Go; Li et al., 1993Go), and that of ChoAS, without any substrate at 1.5 Å resolution (Yue et al., 1999Go), have been determined by X-ray crystallography. Since the amino acid sequence of ChoAS is highly homologous with that of ChoAB (59% identity and 92% similarity), the C{alpha} chains of the two enzymes are almost superimposable. It is expected, therefore, that the native structure of ChoAS is essentially identical with that of ChoAB. However, the crystal structure comparisons between ChoAS and ChoAB reveal significant conformational differences in two loop regions, one of which acts as a lid over the active site and facilitates binding to the substrate and connects to the cofactor, FAD-binding region. Despite the similarities in their primary sequences and three-dimensional structures, the two enzymes have markedly different functions (Noma and Nakayama, 1976Go; Nomura et al., 1995Go; Yue et al., 1999Go). The catalytic efficiency (kcat/Km) was 50-fold higher for the ChoAS than that for ChoAB. The differences in the two loops between the two native structures appeared to translate into differences in substrate activity and specificity (Yue et al., 1999Go). The structural similarities led to the investigation of the reason why ChoAS is a more efficient catalyst than ChoAB. Nonetheless, no obvious differences in the enzyme structures can explain the differences in all of their functions. Knowledge of amino acid sequence homology and three-dimensional structures allows the determination of important roles of specific amino acids residues in the enzyme catalytic mechanism, thermostability, substrate selectivity and structure–function relationships. In describing the high degree of structural similarity between barley ß-glucanase isoenzyme GII and ß-glucanase isoenzyme EII, the differences in substrate specificity and function were achieved without any major changes in the conformation of the polypeptide backbone. Moreover, a relatively small number of amino acid differences in the substrate-binding grooves of the two barley enzymes were found to be responsible for the distinct substrate specificities of the enzymes (Varghese et al., 1994Go).

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., 1997Go). 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., 1997Go; Yamashita et al., 1998Go; Nishiya and Hirayama, 1999Go). The aim was not only to generate a more thermostable ChoAS that could have commercial potential, but also to gain some insight into the structure–function 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., 1997Go), 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., 1998Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents and enzymes

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., 1995Go), 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, 2001Go) 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, 2001Go). 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., 1995Go) 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., 1998Go) 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., 1951Go), using bovine serum albumin as the standard. The purity of each preparation of enzyme was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on a gel that contained 10% (w/v) acrylamide, as described previously (Laemmli, 1970Go), 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., 1974Go). Solutions of enzyme (0.5–1.0 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 Michaelis–Menten 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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 References
 
Construction and activities of mutant derivatives of ChoA

The amino acid sequence identity between the enzyme from Streptomyces (ChoAS) and that from Brevibacterium (ChoAB) is 59% (Figure 2Go). 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 3Go). 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., 1999Go), 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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Fig. 2. Amino acid sequences of cholesterol oxidases from Streptomyces (ChoAS) and Brevibacterium (ChoAB) are aligned. Asterisks and dots indicate identical and related amino acids, respectively.

 


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Fig. 3. Stereoscopic views of the three-dimensional structure of ChoAS. Six sites of mutation, namely L117, L119, V145, Q286, P357 and S379, and both dehydroisoandrosterone (AND) and FAD are shown. The structure was generated using the WebLab Viewer program (Accelrys, San Diego, CA).

 
Purification of wild-type and mutant forms of ChoAS

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 SDS–PAGE analysis. Each preparation of purified enzyme yielded a single band with the same mobility as that of the wild-type enzyme (Figure 4Go). 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., 1995Go).



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Fig. 4. Purification of recombinant wild-type and mutant cholesterol oxidases from E.coli. Samples (1 µg of each purified enzyme) were analyzed by SDS–PAGE (10% acrylamide). M.W. indicates marker proteins.

 
Substrate specificities of wild-type and mutant enzymes

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 IGo). 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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Table I. Substrate specificities of wild-type and mutant cholesterol oxidases
 
Kinetic properties of mutant enzymes

The kinetic parameters for the recombinant wild-type and mutant ChoAS were determined at 37°C using 21–670 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., 1999Go). 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., 1997Go; Sampson et al., 1998Go). 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 IIGo). 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 IIGo).


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Table II. Kinetic parameters of wild-type and mutant cholesterol oxidases with cholesterol and pregnenolone as substratesa
 
To evaluate the role of V145 and S379 residues in the reaction and characterize the mutant enzymes in further detail, as the specificities have been altered, we replaced this Val residue by Asp and Glu in terms of corresponding polarity and this Ser residue by Ala, Val, Arg and Asp in terms of size and/or polarity. The six mutant choA genes encoding enzymes with the V145D, V145E, S379A, S379V, S379R and S379D mutations were confirmed based on their nucleotide sequences. They were expressed in E.coli and the mutant enzymes were purified using cholesterol affinity chromatography and hydrophobic column chromatography as described above. The S379R and S379D mutant enzymes did not have any activities for cholesterol and pregnenolone, nor did they bind to cholesterol in the affinity step of purification. The four mutant enzymes purified had a molecular mass of ~55 kDa as determined by SDS–PAGE (Figure 4Go, S379A and S379V; data not shown). The kcat/Km values of the mutant S379A and S379V enzymes for cholesterol and pregnenolone were similar to that of the wild-type enzyme, whereas those of V145D mutant enzyme for the same substrates were 71- and 14-fold lower, respectively, than that of the wild-type enzyme (Table IIGo). The very low catalytic efficiencies of V145D for cholesterol and pregnenolone resulted from a lower kcat value and a high Km value, while the elevated catalytic efficiency of S379T for pregnenolone was the result of a slightly higher kcat value and a lower Km value than those of the wild-type enzyme.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we constructed 13 mutant enzymes with mutations at six amino acid positions by site-directed mutagenesis. Since conserved amino acid residues are assumed to be important for enzymatic activity and appropriate to targets for mutagenesis, we chose amino acid residues in ChoAS that are not identical with those in ChoAB in the conserved amino acid regions for substitution. Most of the mutant enzymes generated had more or less altered activities or kinetic parameters for cholesterol as compared with the wild-type enzyme. In particular, the mutant L117P enzyme exhibited no activity and the mutant V145D enzyme exhibited a reduced catalytic efficiency for both cholesterol and pregnenolone. However, one of mutant enzymes, S379T, had high catalytic efficiency for cholesterol and pregnenolone.

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., 1998Go). 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., 1994Go). 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., 1997Go; Murooka et al., 1998Go). 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, 1999Go). 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 2Go). 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., 1997Go). 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 5Go). 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 IIGo). 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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Fig. 5. Stereoscopic views of the molecular surface of the amino acid residues around the S379 and the substrate (AND) in ChoAS, colored according to elements (gray, carbon; blue, nitrogen; red, oxygen), are shown. An arrowhead indicates the large cavity around S379. The structures were generated using the WebLab Viewer program (Accelrys).

 
Finally, a more detailed characterization of cholesterol oxidase must await crystallographic information regarding the open wild-type and the mutant enzymes. However, amino acid sequence homology provided direct and important information that allowed the prediction of the functions of some important amino acid residues. Our results showed that genetic engineering approaches could be utilized to identify amino acid residues that confer extreme thermostability and high catalytic activity of cholesterol oxidase enzyme. The V145 residue appears to influence the thermostability and substrate specificity and the S379T mutant has enhanced kcat/Km for cholesterol and pregnenolone with the same thermal stability as the wild-type enzyme (Murooka et al., 1998Go). Genetic engineering altered the active site to accommodate pregnenolone better as the substrate. Cholesterol oxidase was first isolated for use in serum cholesterol assays and subsequently was found to have larvicidal properties (Purcell et al., 1993Go). It is also used as a probe of cholesterol in membrane structure (Lange, 1992Go). These alterations of the catalytic properties in this study are particularly of importance in engineering the protein to be a better diagnostic determinant and larvicide.


    Notes
 
3 Present address: Center of Microbiology and Virology, Polish Academy of Sciences, µodowa St. 105, 93-232 µódz, Poland Back

2 To whom correspondence should be addressed. E-mail: yamashita{at}bio.eng.osaka-u.ac.jp Back


    Acknowledgments
 
We thank Dr Alice Vrielink for her valuable advice and stimulating discussions. A.Z. was supported by a Postdoctoral Fellowship for Foreign Researchers in the Program of the Japan Society for the Promotion of Science (98313).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received September 25, 2001; revised February 19, 2002; accepted February 20, 2002.





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