Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA
1 To whom correspondence should be addressed. E-mail: nicole.sampson{at}stonybrook.edu
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Abstract |
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Keywords: library/mutagenesis/selection/sterol/substrate recognition
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Introduction |
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Cholesterol oxidase heterologously expressed in Escherichia coli is a monomer of 525 amino acids after processing of the signal peptide (Nomura et al., 1995). Despite several X-ray crystal structure determinations of cholesterol oxidase, the structure of the active complex with a full-length sterol is still not known. It is clear that a conformational change must occur to allow entry of the steroid into the binding cavity and to accommodate the C-17 tail of the substrate. The binding cavity contains 14 ordered water molecules in the absence of steroid. Upon steroid binding, at least 13 and most likely all 14 waters are displaced. The structure raises the question as to whether binding energy comes from specific interactions between the substrate and enzyme or whether the affinity of binding is due to the energy released when water is displaced (melted) into bulk solvent (Blow et al., 1997
). If specific binding interactions predominate, then alterations of packing interactions should lead to alterations in substrate specificity. If release of water is the driving force, then differences in binding affinity would only be observed when water is incompletely displaced, for example, by a smaller substrate. An earlier mutagenesis study highlighted the importance of residues 7983 for binding substrate as opposed to binding vesicle (Sampson et al., 1998
). Deletion of the residues resulted in a mutant enzyme that showed improved specificity for dehydroepiandrosterone, although it was still more active with cholesterol. However, this experiment did not distinguish between loss of packing interactions and loss of ordered waters in the binding site.
To test whether it is possible to alter substrate specificity for similarly sized sterols, we chose to screen for mutants that showed a difference in specificity for ß-sitosterol or stigmasterol versus cholesterol. We chose these sterols because they all have extended C-17 tails and intact ring systems (Scheme 2). The ß-sitosterol and stigmasterol have ethyl groups at C-24 that alter the shape of the tail and should allow discrimination on the basis of side-chain packing with the protein, if packing interactions are important in the transition state of binding. Moreover, the toxicity observed with wild-type cholesterol oxidase expression in plants could potentially be ameliorated by introduction of an enzyme with higher specificity for cholesterol over phytosterols, i.e. by introducing orthogonality into substrate recognition.
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Materials and methods |
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Cholesterol was purchased from Sigma Chemical (St Louis, MO), ß-sitosterol and stigmasterol from Steraloids Inc. (Newport, RI) and phospholipids (DOPC) from Avanti Polar Lipids (Alabaster, AL). The plasmid for heterologous expression of Streptomyces sp. cholesterol oxidase, pCO117, was a generous gift from Y.Murooka (Nomura et al., 1995). Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase and alkaline phosphatase were purchased from New England Biolabs (Beverly, MA). Oligonucleotides were obtained from IDT (Coralville, IA). For site-directed mutagenesis, a QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) was used following the manufacturer's instructions. For DNA sequencing, a ABI PRISM dye terminator cycle sequencing kit with AmpliTaq DNA polymeraseFS from Perkin-Elmer (Foster City, CA) was used according to the manufacturer's instructions. Escherichia coli strain XL1 Blue was used for plasmid construction and library amplification and strain BL21(DE3)plysS was used for protein expression and purification. Unless specified otherwise, all chemicals and solvents, of reagent or HPLC grade, were supplied by Fisher Scientific (Pittsburgh, PA). Water for assays and chromatography was distilled, followed by passage through a Barnstead NANOpure filtration system to give a resistivity better than 18 M
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The buffers used were the following: A, 50 mM sodium phosphate, pH 7.0; B, buffer A + 0.020% BSA (w/v); C, buffer A + 2 M (NH4)2SO4.
Cholesterol/LB agar plates were prepared with 0.1% (w/v) sterol, 2% (v/v) 2-propanol and 0.1% (w/v) Triton X-100. Sterol and Triton X-100 were dissolved in 2-propanol. The mixture was added to autoclaved LB/agar solution at 50°C with vigorous stirring. If necessary, the resulting suspension was subjected to sonication to make the suspension uniform. Then the mixture was used to pour the cholesterol/LB agar plates.
Plasmid construction of ChoA mutant libraries
Methods for plasmid preparation were standard unless described otherwise (Sambrook and Russell, 2001). A plasmid, pCO240, that has 10 more unique restriction sites than the parent plasmid pCO202 for wild-type cholesterol oxidase (Sampson et al., 1998
) was constructed by cassette mutagenesis. A 210 bp cassette was constructed by extension PCR. Primers 1 and 2 (50 pmol) had complementary overlapping ends. The primers were denatured at 95°C for 1 min, annealed at 65°C and extended at 72°C for 2 min with Taq polymerase for a total of five cycles. This intermediate cassette was then further extended. Primers 2 and 3 (70 pmol) were mixed with one-tenth of the previous reaction mixture as a template and the mixture was submitted to 10 cycles of PCR, again using Taq polymerase. The cassette was purified and digested with SphI and Eco47III and the digested cassette ligated with similarly digested pCO202. The resulting plasmid pCO240 was used for all subsequent library construction. For pCO261 (
M58), an additional restriction site, MfeI, was introduced by silent mutation of the G57 codon from GGC to GGA to construct plasmid pCO241. The mutation was introduced by PCR mutagenesis using primers 4 and 5. For pCO264 (
F433), pCO242 was constructed by site-directed mutagenesis of pCO240 to remove one of two AgeI restriction sites by replacing the T18 codon ACC with ACA. The mutation was introduced by PCR mutagenesis using primers 6 and 7.
Four libraries that encode one or two mutant amino acids, pCO261 (5M8), pCO262 (
L82/
V85), pCO263 (
M365) and pCO264 (
F433), were constructed by cassette mutagenesis with the primers listed in Table I. Each codon corresponding to the targeted amino acid was replaced with NYS (N = A, C, G, T; Y = C, Y; S = C, G). NYS encodes residues Ala, Val, Leu, Ile, Pro, Met, Ser, Thr and Phe with 16 possible codons. This codon usage limited the amino acids to predominantly hydrophobic residues and reduced the library size. A double-stranded DNA cassette was synthesized from two complementary primers that spanned the nearby unique restriction sites in pCO240, pCO241 or pCO242. For example, pCO261 (
M58) was constructed by cassette mutagenesis in the following way. A double-stranded DNA cassette was synthesized from two complementary primers (primer 8 and 9, Table I) that spanned the MfeI and BspEI restriction sites. The primers introduced NYS (N = A, C, G, T; Y = C, T; S = C, G) at the M58 codon and removed a SphI site by replacing the G57 codon, GGC, with GGA (Gly). Primers 8 and 9 (200 pmol) were phosphorylated with T4 polynucleotide kinase (10 U) for 1 h at 37°C and the enzyme was heat inactivated at 70°C for 10 min. Then the two primers were mixed and denatured at 95°C for 2 min and cooled gradually to room temperature over 3 h. The vector pCO241 was digested with MfeI and BspEI and the annealed cassette was ligated into the purified vector. The ligation mixture was desalted, transformed into XL1 Blue competent (or electrocompetent) cells and plated on to LB/Amp agar plates to generate the library. The colonies on the plates were collected and combined. The level of mutation in the library was checked by restriction with the deleted restriction marker, SphI. The pCO262 (
L82/
V85) library was constructed from pCO240 with the primers 10 and 11 that span the AvrII and ClaI restriction sites. No restriction marker was used because there were no possible silent mutation sites between the AvrII and ClaI sites. The pCO263 (
M365) library was constructed from pCO240 with primers 12 and 13 that span the restriction sites MluI and NruI and introduced an XbaI site by replacing the G368/L369 codons, GGC/CTG, with GGT/CTA (GlyLeu). The pCO264 (
F433) library was constructed from pCO242 by cassette mutagenesis with primers 14 and 15 that introduced a KpnI site by replacing the G434 codon, GGC, with GGT (Gly) and ligated into AgeIStuI restricted vector.
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Synthesis of lipid vesicles
Small unilamellar vesicles (SUV) were made by ethanol injection from mixtures of steroid and lipids (Kremer et al., 1977). Cholesterol, ß-sitosterol or stigmasterol were used for the activity assay. Lipid DOPC (16.7 µmol) was mixed with steroids (5.55 µmol) as a CHCl3 solution in a round-bottomed flask, dried as a thin film under reduced pressure in a rotary evaporator for 30 min and dried under high vacuum for 2 h. The resulting thin film was resuspended in 5 ml of ethanol. The mixture was rapidly injected into 150 ml of buffer A with vigorous stirring.
Medium, 100 nm unilamellar vesicles (MUV) were made from mixtures of DOPC by extrusion (Hope et al., 1985). Lipids (33 µmol) were mixed as CHCl3 solutions in a round-bottom flask, dried as a thin film under reduced pressure in a rotary evaporator for 20 min and evacuated under high vacuum for 2 h. The lipids were resuspended in 10 ml of buffer A with vortex mixing. Five freezethaw cycles, at 80 and 37°C, followed by 10 extrusion cycles through two stacked 100 nm filters (Costar, Cambridge, MA) using a nitrogen gas pressure of 350400 psi provided a homogeneous batch of vesicles. Phospholipid concentrations of vesicles were measured using the Stewart assay (Stewart, 1959
). Sterol concentrations were measured by lysing vesicles with 0.1% Triton X-100 and using cholesterol oxidase to quantitate total sterol concentration as described below.
Screening of cholesterol oxidase library with three sterol substrates
A portion of our constructed library (pCO261 to pCO266) was transformed into XL1 Blue and plated on to LB/Amp plates. Individual colonies were isolated and cultured in 1.5 ml of 2x TY/amp media in each well of a 96-well plate. The cultures were grown for 10 h at 37°C. An aliquot (200 µl) of the cell culture was removed and pelleted for future plasmid preparation. The remaining cell cultures were induced with 0.4 mM IPTG for another 10 h at 30°C. The cells were collected by centrifugation, frozen and lysed with 50 µl of Bugbuster (Novagen, Madison, WI). Buffer A (400 µl) was added and the lysate centrifuged and stored for screening.
The amount of total protein in the supernatant was determined by Bradford assay (Bradford, 1976). The assay mixture consisted of 195 µl of SUV [DOPC:sterol = 3:1 (mol/mol)] and 5 µl of the crude lysate supernatant with a final DOPC concentration of 105 µM. The presence of 2.5% Bugbuster that contains detergent did not cause lysis of SUVs as judged by light scattering. The activity of cholesterol oxidase towards different sterols was measured by following the appearance of the product, conjugated enone at 240 µm (
240 = 12 100 M1 cm1) at 37°C. The slope of the first 10% of the reaction was determined by linear regression and converted to specific activity relative to the total protein amount. The specific activity of different mutants was compared with the wild-type supernatant from pCO240 expressed in a similar fashion.
Purification of mutant cholesterol oxidases
Cell paste of E.coli BL21(DE3)plysS(pCO202) (Sampson et al., 1998) was obtained from 1 l of 2x YTampicillin (200 µg/ml) medium grown at 28°C for 8 h after addition of IPTG (100 µg/ml) at A600 = 0.8 by centrifugation at 4000 g for 30 min. The pellet was resuspended in 20 ml of buffer A and lysed by French press at 18 000 psi. All subsequent steps for the mutant cholesterol oxidases were conducted at 4°C. Cell debris was removed by centrifugation at 135 000 g for 120 min. The supernatant was precipitated with 1.0 M (NH4)2SO4 and the pellet was discarded. (NH4)2SO4 was added to the supernatant to a final concentration of 2.5 M. The pellet was obtained by centrifugation at 4000 g. This pellet was resuspended in buffer A (5 ml) and desalted using dialysis (MWCO 60008000) against buffer A. The dialysate was loaded on to a column of DEAE-cellulose (30 mm x 25 cm, DE-52, Whatman) pre-equilibrated with buffer A. Fractions were collected by elution with buffer A (100 ml). Typically, 15 ml fractions were collected; however, fractions that appeared yellow in color were limited to 7.5 ml. Fractions containing cholesterol oxidase (as determined by SDSPAGE) were combined and concentrated by (NH4)2SO4 precipitation (3.0 M). The pellet was dissolved in buffer A to give a final concentration between 10 and 20 mg/ml of protein and (NH4)2SO4 was slowly added until the solution turned slightly turbid (typically, 2.5 M). The precipitate formed was pelleted by centrifugation and the clarified supernatant was transferred to a fresh container, where it was allowed to crystallize over 2 days at 4°C. The microcrystalline protein was collected by centrifugation, dissolved in buffer A and ultrafiltered (YM30 membrane, Amicon, Danvers, MA) into buffer A. The protein was further purified on a butyl-Sepharose column (30 ml butyl-Sepharose-4 Fast Flow, XK 16/40, Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with buffer C. The protein was eluted by running a linear gradient from 100% buffer C to 100% buffer A (270 ml). Fractions (4.5 ml) were collected and the elution profile was monitored at A280. Fractions were assayed for content and purity by SDSPAGE. Fractions containing pure cholesterol oxidase (>98%) were combined and ultrafiltered (YM30 membrane) into buffer A. Typically 3040 mg of mutant cholesterol oxidase were obtained per liter of culture.
Steady-state fluorescence binding measurements
Binding of cholesterol oxidase to MUVs was assayed through the quenching of intrinsic tryptophan fluorescence. All binding assays were conducted in buffer A at 25°C. Tryptophan was excited at 280 nm and spectra were collected from 310 to 450 nm. A 310 nm cutoff filter was placed before the emission monochromator to reduce scattered light. Sample signals were corrected for light fluctuation by simultaneously monitoring the exciting light on a reference photomultiplier. Protein (510 µg/ml) was titrated with increasing amounts of lipid vesicles (0100 µl of a 3.3 mM stock solution) to a final concentration of 500 µM. Emission was corrected for any background signal by performing a titration in the absence of protein.
Binding constants were analyzed by first correcting spectra for dilution and background signal. The spectra were integrated and normalized to their value in the absence of added lipid. The adsorption isotherm was fitted to the hyperbolic Equation 1 using KaleidaGraph (Synergy Software, Reading, PA):
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Steady-state enzyme kinetics
Stock solutions of sterol SUV were prepared in buffer A by ethanol injection as described above. Dilute enzyme stock solutions were prepared in buffer B. Initial velocities were measured by following the formation of conjugated enone as a function of time at 240 nm (240 = 12 100 M1 cm1) (Smith and Brooks, 1977
). Independent sets of data were fit to the hyperbolic Equation 2 using Kaleidagraph:
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All molecular mechanics and dynamics was performed with InsightII v. 95.0 (Biosym Technologies) using the Amber forcefield version 1.5 and Discover version 2.97.
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Results |
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In the absence of an open X-ray crystal structure of cholesterol oxidase with cholesterol or cholesterol analog bound, molecular mechanics and dynamics were used to model a structure. Dehydroepiandrosterone was first modeled into the active site of Streptomyces cholesterol oxidase (1b4v.pdb) using the structure of the Brevibacterium enzyme that is 58% identical in amino acid sequence and nearly identical in three-dimensional structure (Yue et al., 1999). The Brevibacterium cholesterol oxidasedehydroepiandrosterone structure (1coy.pdb) was superimposed on the Streptomyces structure by aligning FAD, His447, Asn485 and Wat541. The isoprenyl tail of cholesterol was then built on to the dehydroepiandrosterone backbone. Addition of these eight carbons caused severe steric clashes between loop 7386 and the steroid. Residues 73 and 86 were chosen as hinge points to rotate the loop rigidly into an open conformation. The loop was opened by changing
for C73N74 from 175° to 170° and
for Asn86 from 69° to 130° and minimizing the structure with steepest descent mechanics to restore bond lengths and angles to equilibrium positions. This structure was used as the starting point for molecular dynamics calculations to search for the lowest energy conformation. The dynamics were performed on the residues within 20 Å of the substrate. The cholesterol O3 atom was not allowed to move. Since only residues within 20 Å of the steroid were included in the calculation, a contiguous single polypeptide was not present in the calculation. The loop terminal residues Lys69, Ile89, Tyr428 and Ala434 were tethered and the residues more than 15 Å from the cholesterol tail were constrained, that is, their atomic positions were fixed during the dynamics calculations. In this open model, the
-helix that forms part of the lid was maintained (Figure 1A). Inspection of the model identified five hydrophobic side chains that may contact C24C27 of cholesterol: Met58, Leu82, Val85, Met365 and Phe433 (Figure 1B and C). These residues face inwards and therefore any activity changes arising from mutations at these positions should result from changes in steroid binding rather than binding to the lipid bilayer surface.
Library construction
Seven libraries were constructed by cassette mutagenesis. Some of these constructions required the creation of wild-type plasmids that contained additional restriction sites for manipulation. A cassette encoding silent mutations that introduced ten restriction sites between codons 58 and 128 was synthesized by extension of overlapping primers to form an intermediate 160 bp cassette. This cassette was further extended by PCR with the original antisense primer 2 and the sense primer 3 to yield a 234 bp cassette that was restriction digested at the 5' and 3' ends with SphI and Eco47III, respectively and inserted in the wild-type cholesterol oxidase plasmid to form pCO240. The restriction sites introduced were: BspMII, SplI, AvrII, HpaI, ClaI, PpuMI, BclI, XcaI, SnaBI and SpeI. Plasmid pCO240 was further modified to insert an Mfe I site to form pCO241 or to remove an AgeI site to form pCO242. For each library, a cassette encoding the relevant residues was synthesized from 5'-phosphorylated primers in which the targeted codons were replaced with NYS. This degenerate codon provided mutants with Ala, Val, Leu, Ile, Met, Pro, Ser, Thr and Phe at the targeted position. Each cassette also inserted or removed a silent, unique restriction marker for verification of mutation. Initially, four libraries were prepared that yielded pCO261 (M58), pCO262 (
L82/V85), pCO263 (
M365) and pCO264 (
F433). These libraries were then combined to yield three more libraries of mutants: pCO265 (
M58/L82/V85), pCO266 (
M365/F433) and pCO267 (
L82/V85/F433). For the single- and double-residue libraries, 1 x 103 and 2 x 103 transformants, respectively, were collected for each library. For the three residue libraries, 2.5 x 104 transformants were collected.
Screening for activity and substrate specificity
A cholesterol agar plate assay was used to screen libraries on a large scale (Nishiya et al., 1998). This assay utilized the different optical properties of cholesterol/Triton X-100 micelles versus cholest-4-en-3-one/Triton X-100 micelles. A 2-propanol solution of cholesterol and Triton X-100 was added with rapid stirring to liquified LB agar to prepare a finely dispersed suspension. Upon solidification, the cholesterol/LB agar plates were opaque. Bacterial colonies producing catalytically active cholesterol oxidase develop halos as a result of oxidation of the cholesterol in the agar. Cholesterol oxidase expression is under control of the tac promoter (derived from pKK223-3) and is incompletely repressed in the XL1Blue (lacI) E.coli strain. Wild-type colonies produced halos in 30 ± 2 h. A catalytically impaired mutant, H447Q, that has a kcat 280-fold lower than wild-type (Kass and Sampson, 1998
), took 60 h to produce a halo. This assay was used to extensively screen all the libraries in order to assess the number of active mutants (Table II). However, the halo effect was much more difficult to discern for ß-sitosterol and stigmasterol and halo formation was insensitive to small changes in catalytic activity. Therefore, a spectrophotometric activity assay was used to screen the activity of a more limited number of cell lysates with all three sterols. Mutant libraries were expressed in a 96-well format and cell lysates assayed with the three substrates presented as vesicles. The lysates were assayed at a single vesicle concentration and the vesicles were 25 mol% sterol mixed with DOPC. Under these conditions, >95% of the wild-type enzyme is bound to the vesicle surface and the enzyme is not saturated with sterol (Ahn and Sampson, 2004
). Moreover, under these conditions, the wild-type kcat*/Km* values for the three sterols are very similar (Table III) and any changes in specificity should be readily detected. We chose a lipid that remains in the liquid-disordered phase upon mixing with sterol to avoid the effects that differential formation of lipid rafts with different sterols might have on activity measurements.
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Characterization of selected mutants
We sequenced a number of active mutants from the M58,
M365 and the
F433 libraries and found that the mutants directly reflected the NYS codon usage and all possible amino acid substitutions were present. We sequenced a small number of mutants from the
L82/V85 and
L82/V85/F433 libraries, 12 and five, respectively, in order to select two for further analysis. Again, the sequences mirrored the diversity of our codon usage. We chose L82S/V85I/F433S and L82A/V85T/F433L for purification and further analysis. Both mutants were heterologously expressed and purified in a manner identical with wild-type cholesterol oxidase.
We measured kcat*/Km* as described previously (Ahn and Sampson, 2004) because with lipid bilayers tested thus far, the wild-type enzyme is not saturable with cholesterol. Moreover, kcat*/Km* is the relevant rate constant for examining substrate specificity. The changes observed were less than 2-fold for both mutants on each of the three sterols tested.
In addition, we measured two dissociation constants. Kapp, the coupled equilibrium constant for enzyme binding to vesicle and substrate binding, was unchanged regardless of sterol tested. Kd, the enzymevesicle dissociation constant measured for non-sterol-containing DOPC vesicles, was also unchanged upon mutation. Hence the results obtained from the screening assay were confirmed. Mutations at five different sites that contact the substrate tail can cause up to 10-fold reductions in catalytic activity, yet have no effect on substrate specificity.
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Discussion |
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We modeled a full-length sterol into the cholesterol oxidase active site of the closed structure. This structure was adjusted to accommodate the cholesterol tail and conformational space searched using molecular dynamics. The structures converged on the model shown in Figure 1. In this model, the loop is more open than we might expect in a final bound structure. However, it allowed us to visualize what residues would come into contact with the last four carbons, C23C27 of the cholesterol tail, upon binding to the enzyme and to target residues that should not come in contact with the lipid bilayer. These residues were targeted for mutagenesis and screening of activity.
We reasoned that only hydrophobic groups would be tolerated as mutated residues and therefore generated seven libraries of random mutants that mutated the targeted five amino acids to alternate hydrophobic groups within the limits of the genetic code. We initially used an agar plate cholesterol activity assay in order to screen the libraries more fully. We then performed activity assays with the three substrates, cholesterol, ß-sitosterol and stigmasterol, in order to obtain more precise data. These assays were performed on smaller subsets of the libraries containing multiple mutation sites. These screens revealed that all members of the libraries retained at least 10% of the activity of wild-type cholesterol oxidase. Moreover, there was no alteration in substrate specificity. That is, when the kcat*/Km* for cholesterol was reduced 10-fold, the kcat*/Km*s for ß-sitosterol and stigmasterol were identically reduced.
We selected two mutants for purification and further kinetic analysis to verify the results of our screen. These mutants were chosen because their sequences showed significant changes from the wild-type sequence. They contained the residues serine and threonine that have hydrogen-bonding hydroxyls in addition to their hydrophobic aliphatic side chains. The presence of the polar residues should not be compatible with hydrophobic packing. In both cases, the kcat*/Km*s were unchanged, as were the binding affinities of the enzymes for the vesicle surface (Kapp and Kd). These results suggested that there are no specific interactions between these amino acid residues and the tail of the sterol.
The targeted residues appear to form a hydrophobic channel that allows sterols to enter the enzyme binding site. The 3-hydroxyl must enter first as the active site is at the bottom of the binding cavity. The sterol AD ring system is relatively more bulky and rigid than the hydrocarbon tails of cholesterol, ß-sitosterol or stigmasterol. Hence the diameter of the hydrophobic channel must be large and flexible enough for the steroid framework to pass through before the channel encounters the tail. Rather than present specific bumps and holes into which the tail meshes, the channel residues may simply provide a greasy, slightly disordered environment around the tail that can accommodate a variety of structures.
Previous substrate specificity engineering experiments by Toyama et al. (2002) targeted residues that were not conserved between the Brevibacterium and Streptomyces cholesterol oxidases. The residues targeted were much closer to the FAD and deeper in the binding site. Although they only screened against truncated steroids, e.g. pregnenolone, and did not screen against sitosterol or stigmasterol, they also only observed parallel changes in catalytic activity with mutation. For example, mutation of Leu119 resulted in a 2-fold decrease in activity for both cholesterol and pregnenolone. Likewise, mutation of Ser379 resulted in an
2-fold increase in activity for both substrates. Hence it appears that mutation of a wide variety of specific amino acid residues can alter the catalytic efficiency of the enzyme without leading to altered substrate specificity. The only reversal of substrate specificity known to date is that which occurred upon deletion of five residues of the enzyme (Sampson et al., 1998
).
Both our experiments and those of Toyama et al. suggest that specific packing interactions contribute only very weakly, if at all, to the binding affinity and specificity of cholesterol oxidase for its substrate. Because the substrate is presented to the enzyme from a hydrophobic milieu, e.g. a lipid bilayer or detergent micelle, the binding energy cannot derive from desolvation of the hydrophobic steroid. The remaining source of binding energy is desolvation of the enzyme active site. This active site contains 14 crystallographically ordered waters. Their high order suggests that upon dissociation from the substrate-binding site, a large amount of energy may be released. It is this release of water that favors binding of the hydrophobic steroid. Therefore, in order to change the substrate specificity, it would be necessary to engineer the site such that there is one more water that can only be displaced by the desired substrate. This will require a much more subtle engineering approach than has been currently utilized in this or other systems.
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Acknowledgments |
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Received January 21, 2004; revised April 29, 2004; accepted April 30, 2004.
Edited by Derek N.Woolfson
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