From the Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Received for publication, March 9, 2001, and in revised form, April 6, 2001
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ABSTRACT |
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Six amino acid locations in the soluble castor
Plant-soluble fatty acid desaturase enzymes introduce a double
bond regiospecifically into a saturated acyl-ACP substrate. The
reaction involves activation of molecular oxygen by a two-electron reduced diiron center coordinated by a four-helix bundle that forms the
core of the desaturase architecture (1, 2). The archetype of this class
is the Several naturally occurring variant desaturase enzymes have been
isolated from tissues that accumulate unusual fatty acids based on
their homology to the archetype
18:0-ACP2 desaturases (5,
8-11). The specific activity profiles of these enzymes are consistent
with a role of producing the corresponding unusual fatty acids.
However, they exhibit very poor specific activities compared with all
stearoyl-ACP desaturases reported to date and have proved ineffective
in producing altered fatty acid phenotypes when heterologously
expressed (Ref. 12 and data not shown). Recent observations from the
Ohlrogge group suggests that vegetative
ferredoxins,3 and specific
ACP isoforms may be required for optimal activity of the variant
desaturases (13, 14). In addition to showing poor activities, variant
desaturases tended to form insoluble aggregates when purified.
Low stability and poor catalytic rates are properties shared by many
newly evolved enzymes that arise as gene duplication events in which
selection for stability and/or turnover is released, while mutations
accumulate that finally result in an alteration of function (15, 16). A
prediction of this model is that it should be possible to redesign
archetypal enzymes that exhibit desired stability and/or turnover to
achieve an altered substrate specificity while retaining the desired
properties of the parental paralog.
Bacterial unsaturated fatty acid auxotrophs have been used for the
isolation and characterization of both membrane-bound and soluble fatty acid desaturase systems (17-19). We recently
described a bacterial selection system capable of distinguishing
between soluble desaturase enzymes with different chain length
specificities (19). Soluble desaturases specific for 18-carbon chain
length are unable to complement MH13, an unsaturated fatty acid
auxotroph of Escherichia coli defective in the anaerobic
dehydratase/isomerase pathway of double bond incorporation into fatty
acids, presumably due to the lack of available 18:0-ACP substrate.
However, desaturase variants capable of recognizing substrates of 16- or 14-carbon chain length are able to complement the unsaturated fatty
acid auxotrophy. To test the selection system, we identified four amino acid positions at the base of the substrate-binding cavity
(114,4 118, 179, and 188) and
constructed libraries of mutants saturated for substitutions at each of
the locations individually. Variants best able to complement the
unsaturated fatty acid auxotrophy were isolated and characterized. The
resulting enzymes, like those derived from rational design efforts,
showed relatively poor specific activity compared with the wt enzyme
with its preferred substrate, 18:0-ACP (19, 20). This result is
consistent with the observation that hydrophobic partitioning plays a
substantial role in the selectivity and reaction rate for the wt castor
However, small changes in relative positioning of substrate relative to
the active site have been shown to exert large effects on catalytic
rates (21, 22). We therefore used combinatorial saturation mutagenesis
at positions known to affect substrate specificity to maximize the
likelihood of identifying variants with desired improvement in turnover
rates. These combinatorial genetic experiments led to the
identification of two key substitutions (T117R/G188L) that when made
alone in the wt castor Random Point Mutagenesis--
The portion of the castor
Saturation Mutagenesis at Codons 117 and
181--
Saturation mutagenesis was performed at codons for amino
acids 117 and 181 of the castor Combinatorial Saturation Mutagenesis--
Overlap extension PCR
was used to construct a library in which combinations of one of 20 possible amino acids were expressed at each of six positions (114, 117, 118, 179, 181, and 188) of the castor Construction of Mutant 5.2 (T117R/G188L) of the Castor
E. coli Unsaturated Fatty Acid Auxotroph
Complementation--
Castor Fatty Acid Analyses--
MH13/pAnFd cells expressing castor
acyl-ACP desaturase variants in vector pLac3 were grown at 30 °C to
A600 Substrate Specificity and Kinetic Analysis of Castor
Choice of Residues for Mutagenesis--
It is difficult to predict
the locations of amino acids that can affect a property of interest
(30). For this reason we performed random mutagenesis of the entire
363-amino acid coding region of the desaturase and subjected the mutant
population to a selection system to identify mutants active on
substrates with chain length <18 carbons. In this way, five positions
(114,4 117, 118, 179, and 181) capable of affecting
chain length specificity were identified (Table
I). Mutagenesis was at, or close to,
saturation based on the isolation of the same amino acid changes
multiple times (data not shown). Three of the amino acid positions
(114, 118, and 179) identified by random mutagenesis were coincident with a group of four (114, 118, 179, and 188) that were previously shown to affect chain length specificity (19), and two new amino acid
locations, 117 and 181, were identified (Fig.
1). In the case of this soluble
desaturase enzyme, all six amino acid positions cluster to the region
adjacent to the bottom of the substrate-binding pocket, and no sites
remote from the substrate binding site were identified. These six
locations were therefore selected as targets for combinatorial
mutagenesis.
Comparison of the activities of point mutants at each of the selected
sites with those from saturation mutagenesis at the same location
showed that for four of the six sites (114, 117, 179, and 181) superior
mutants were obtained by saturation mutagenesis, an equal improvement
for the fifth site (118), and no point mutation was obtained for the
sixth site (188) (Table I). The observation that saturation mutagenesis
at single sites resulted in larger improvement in the property of
interest compared with point mutagenesis prompted us to ask if even
larger improvements in specific activity for substrates with fewer than
18 carbons could be made by simultaneously randomizing the six
positions able to affect substrate specificity.
Combinatorial Saturation Mutagenesis--
A library of variants
randomized for amino acid substitutions at all six sites was
constructed by overlap extension PCR. To select for improvement in
activity with substrates <18 carbons in length, the library was
introduced into the unsaturated fatty acid auxotroph and challenged on
media lacking unsaturated fatty acids.
Nineteen colonies that exhibited strong growth under selective
conditions subjected to both sequence and activity analyses. Sequence
analysis revealed that all 19 clones contained a unique combination of
amino acids at the six randomized positions. When analyzed for specific
activity, com2 (M114A/T117R/L118G/P179V/T181V/G188L) showed the
greatest improvement in substrates containing fewer than
18-carbons, i.e. 52-fold for 14-carbon substrates and
15-fold for 16-carbon substrates (Table
II). Of note, five of the 19 strongly complementing mutants (com2, com3, com4, com9, and com10) contained the
same pair of changes, T117R/G188L, found in com2 (Table II). The wide
range of specific activities among the different complementing mutants
containing the T117R/G188L pair of mutations suggested the presence of
detrimental mutations in one or more of the remaining four positions.
This was confirmed by construction of the double mutant (5.2)
T117R/G188L (in which the other targeted positions contained residues
found in wt desaturase) that showed a 35-fold increase in specific
activity with respect to the 16-carbon substrate and a 24-fold increase
for the 14-carbon substrate (Table II).
Kinetic Analysis of wt and Mutant 5.2--
To gain insight into
the mechanism underlying changes in substrate specificity, we performed
a comparative kinetic analysis on wt and mutant 5.2 (Fig.
2 and Table
III). The data show that wt desaturase
has a different Km for different substrates, 0.46 µM for 18-carbon, 5.0 µM for
16-carbon, and 4.6 µM for 14-carbon substrates.
However, in the case of the double mutant 5.2 the Km
for 16-carbon substrate has decreased by an order of magnitude from 5.0 to 0.55 µM, while the Km for
18- and 14-carbon substrates have changed from 0.46 to 0.98 µM and from 4.6 to 1.1 µM,
respectively. Together these changes result in an increase in
specificity factor from 0.56 to 46 µmol Regiospecificity--
Selection for one parameter can lead to
undesirable changes in unrelated parameters (31). For the desaturase
changes in regiospecificity had accompanied earlier attempts to alter
chain length specificity (19). We therefore determined the position of
the introduced double bond by gas chromatography-mass
spectrometry analysis of the dimethyl disulfide derivative of
the desaturated fatty acid product of mutant 5.2. The only detectable
product was 16:1 in which the double bond was exclusively located at
the Combinatorial saturation mutagenesis of six residues shown
individually to affect chain length specificity, in combination with
selection using an unsaturated fatty acid auxotrophy complementation system in combination with logical redesign, led to the engineering of
a desaturase enzyme with preference for 16-carbon substrate with
kinetic parameters similar to those of wt desaturase for the 18-carbon
substrate. The activity of the resulting mutant 5.2 is >100-fold
improved with respect to those reported for naturally occurring
16:0- To understand the mechanism underlying the observed improvement in
specific activity of mutant 5.2, we performed a kinetic analysis in
which we included wt castor The Km value for mutant 5.2 with the 16-carbon
substrate decreased ~10-fold with respect to that of the wt enzyme, i.e. to 0.55 (±0./06) µM from 5.0 (±0.56)
µM, respectively. It is interesting to note that the
physiological selection system used to identify the mutations used to
engineer mutant 5.2 resulted in an enzyme with a Km
value almost identical to the Km of 0.46 (±0.06)
µM reported (above) for the wt enzyme with the 18-carbon substrate.
In addition to changes in binding affinities for different substrates
between wt and mutant 5.2, there were also substantial changes in
kcat. Mutant 5.2 showed a large increase in
kcat of 25.3 min9-18:0-acyl carrier protein (ACP)
desaturase were identified that can affect substrate specificity.
Combinatorial saturation mutagenesis of these six amino acids, in
conjunction with selection, using an unsaturated fatty acid auxotroph
system, led to the isolation of variants with up to 15-fold increased
specific activity toward 16-carbon substrates. The most improved
mutant, com2, contained two substitutions (T117R/G188L) common to five
of the 19 complementing variants subjected to further analysis. These
changes, when engineered into otherwise wild-type 18:0-ACP desaturase
to make mutant 5.2, produced a 35-fold increase in specific activity
with respect to 16-carbon substrates. Kinetic analysis revealed changes
in both kcat and Km that
result in an 82-fold improvement in specificity factor for 16-carbon
substrate compared with wild-type enzyme. Improved substrate
orientation apparently compensated for loss of binding energy that
results from the loss of desolvation energy for 16-carbon substrates.
Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of
magnitude higher than those of known natural 16-carbon specific
desaturases. These data support the hypothesis that it should be
possible to reengineer archetypal enzymes to achieve substrate
specificities characteristic of recently evolved enzymes while
retaining the desired stability and/or turnover characteristics of a
parental paralog.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9-18:01-ACP desaturase
required by all plants for the maintenance of membrane fluidity (2, 3).
While this enzyme primarily desaturates stearoyl-ACP, it is also active
to a minor extent with palmitoyl-ACP (4, 5). The crystal structure of
the native desaturase revealed a pocket capable of accommodating an
18-carbon substrate in the extended conformation adjacent to the diiron
active site (6). While a pocket capable of binding 18-carbon substrates
could also structurally accommodate 16- and 14-carbon substrates,
reduced turnover with 16- and 14-carbon substrates has been correlated with a loss of binding energy corresponding to the desolvation of two
or four fewer methylene groups (7).
9-18:0-ACP desaturase (7). This conclusion, along with
the results of our previous efforts in producing enzymes with desired
specificity but poor turnover rates, suggested that this
physico-chemical parameter might place an upper boundary to the
reaction rate of desaturase with the desired 16-carbon substrate and
that it might not be possible to engineer a
16:0-
9-desaturase with catalytic properties similar to
those of the wt enzyme with its preferred substrate.
9-18:0-ACP desaturase background
yielded a
9-16:0-ACP desaturase with a turnover rate
approaching that of wt enzyme with 18:0-ACP. This improvement resulted
from an increase in kcat and decrease in
Km that yielded an improvement in specificity factor
for 16-carbon substrate of 82-fold with respect to wt enzyme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9-18:0-ACP desaturase corresponding to the mature
polypeptide was excised from the clone pRCD1 (3) with the use of
EcoRI and XbaI. The 1683-base pair
fragment was subject to partial digestion with DNase I (Sigma) and the
resulting fragments separated by agarose gel electrophoresis. Fragments ranging in size between 100 and 250 base pairs were excised and subjected to primerless PCR to facilitate assembly of the full-sized gene along with the incorporating point mutations (23). The authentic
sized product was excised from an agarose gel and amplified by PCR
using primers: 5'-CACACAGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA-3' (Primer
A) and 5'-GTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCG-3' (Primer B). The
amplified fragment was restricted with XbaI and
EcoRI and cloned into the corresponding sites of pLac3. The
resulting library of mutants was transformed into E. coli MH13/pAnFd and the cells challenged to growth in the absence
of unsaturated fatty acids.
9-18:0-ACP desaturase by
replacing the target codon by NNN with the use of overlap-extension PCR
(24) as described previously (19). The mature castor
9-18:0-ACP desaturase was used as the template, and
amplification reactions were conducted with Pfu polymerase
(Stratagene). Saturating mutations were introduced at the codon for
amino acid 117 by initially conducting two separate amplification
reactions using the following oligonucleotide primer combinations:
Reaction 1-Primer A and
5'-CCAAATTGCCCAAGACGTCGGACTTGCACCTGTTTCATCCCGAACTCCATCCAANNNATTCAGCATTGTTTG-3' (Primer C) and Reaction 2-R5'-GAAACAGGTGCAAGTCCGACGTCTTGGGCAA-3' (Primer D) and 5'GTTTTCTGTCCGCGGATCCATTCCTG3' (Primer E). Agarose gel-purified reaction products were combined and amplified together with primer A and Primer E. The resulting product was digested with
XbaI and SacII and ligated in place of the
corresponding portion of the coding sequence for the mature wild-type
castor
9-18:0-ACP desaturase in pLac3d. Saturation
mutagenesis at codon 181 was achieved by amplification of the castor
desaturase with: 5'-GGTTCAGGAATGATCCGCGGNNNGAAAAGAGTCCATACC-3' (Primer
F) and 5'-GCAAAAGCCAAAACGGTACCATCAGGATCA-3' (Primer G). The resulting
fragment was restricted with BamHI and Acc65I and
cloned into the corresponding portion of the coding sequence for the
mature wild-type castor
9-18:0-ACP desaturase in pLac3d.
9-18:0-ACP
desaturase simultaneously. The codon NNK(G/T) was introduced at each of
these targeted positions, because it has 32-fold degeneracy rather than
the 64-fold degeneracy of NNN, and it encodes all 20 amino acids
without rare codons. Three independent PCR amplification reactions of
the wild-type castor
9-18:0-ACP desaturase were
performed using the following oligonucleotide primer combinations:
Reaction 1-Primer A and 5'-TTGATAAGTGGGAAGGGCTTCTTCCGTT-3' (Primer H), Reaction
2-R5'- AACGGAAGAAGCCCTTCCCACTTATCAAACANNKCTGAATNN- KNNKGATGGAGTTCGGGATGAAAC-3'
(Primer I), and 5'-TCCATTCCTGAACCAATCAAATATTG-3' (Primer J), Reaction
3-R5'-TTGATTGGTTCAGGAATGGATNNKCGGNNKGAAAACAGTCCATACCTTNNKTTCATCTATACATCATTCC-3' and Primer G. The three agarose gel-purified reaction products were
combined and amplified together with Primer A and Primer G. The
resulting product was digested with XbaI and
Acc65I and ligated in place of the corresponding portion of
the coding sequence for the mature wild-type castor
9-18:0-ACP DES in pLac3d. The library contained
>106 independent clones. A sample of 19 desaturase clones
isolated from colonies that exhibited strong growth in the
complementation assay were sequenced. All showed unique sequence
combinations at the six targeted positions.
9-18:0-ACP Desaturase--
Mutant 5.2 was constructed
by ligation of the KpnI-BamHI fragment from
mutant T117R into the corresponding region of mutant G188L.
9-18:0-ACP desaturase
variants in plasmid pLac3d were transformed into E. coli
MH13/pAnFd cells (19). Cells were grown on LB plates that contained
ampicillin (100 µg/ml), chloramphenicol (35 µg/ml), and kanamycin
(40 µg/ml) (for selection of pLac-des, pLacAnFd, and the MH13 strain,
respectively). For permissive growth, plates were supplemented with
oleic acid and Tergitol NP-40 (Sigma) at final concentrations of 250 µg/ml and 0.2% (v/v), respectively. Selection for complementation
was performed on agar plates containing 0.4 mM
isopropyl-
-D-thiogalactopyranoside (lacking unsaturated fatty acid) incubated at 30 °C. Complementation was confirmed by growth of selected colonies in liquid media of the same
composition at the same temperature. The sequences of the selected
variants of the castor
9-18:0-ACP desaturase were
determined to identify the substitution(s).
0.3 to 0.5 in 25 ml of LB media
supplemented with chloramphenicol, kanamycin, ampicillin, and
isopropyl-
-D-thiogalactopyranoside as described above.
Cells were collected by centrifugation and lipids extracted and dried
under N2 (25). Fatty acid methyl esters were made by
transesterification with sodium methoxide (26) and analyzed with the
use of a Hewlett-Packard 6890 GC fitted with a 30 m × 0.25 mm
(inner diameter) HP-INNOWAX (Hewlett-Packard) column. Oven
temperature was raised from 170 °C at 3.5 °C/min to 185 °C after a 25-min hold. Double bond positions of monounsaturated fatty
acid methyl esters were located by gas chromatography-mass spectrometry of dimethyl disulfide derivatives (27).
9-18:0-ACP Desaturase Variants--
Desaturase variant
open reading frames were transferred from pLac3d to pET9d. Recombinant
protein was generated by expression in E. coli BL21(DE3)
cells and enriched to 90-95% purity by 20CM cation exchange
chromatography (Applied Biosystems). Castor
9-18:0-ACP
desaturase variants were assayed with [1-14C]14:0-,
[1-14C]16:0-, or [1-14C]18:0-ACP substrates
with the use of recombinant spinach ACP-I (28). Methyl esters of fatty
acids were analyzed by argentation TLC and radioactivity in products
quantified as described previously (11, 18). Wild type castor
9-18:0-ACP desaturase and mutant 5.2 assays were
performed in triplicate for each of five concentrations typically
ranging from one-fifth to five times the Km (where
practical) and with each of three substrates: 14:0-, 16:0- and
18:0-ACP. Data were processed with the use of the GraFit data analysis
and graphics suite version 3.01 (29). Kinetic parameters and statistics
were based on preprogrammed algorithms within the GraFit suite.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Specific activity of various single-site mutants of the castor
9-18:0-ACP desaturase relative to wt
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Fig. 1.
Location of amino acids selected for
combinatorial saturation mutagenesis. A stearic acid moiety
shown in green is modeled into the active site, iron ions
are shown in magenta, helices and loops are shown
(gray behind the plane of the fatty acid and
yellow in front). The six residues subjected to mutagenesis
are indicated in cyan, and the modeled T117R/G188L
substitutions in mutant 5.2 are shown in violet. Atom
colors: gray, carbon; blue, nitrogen;
red, oxygen; and green, sulfur.
Substitutions at specific positions of the castor 9-18:0-ACP
desaturase mutants containing the T117R/G188L pair of mutations and
their specific activity relative to wt
1
min
1 for the 16-carbon substrate, and a
decrease from 92 to 5 µmol
1
min
1 for the 18-carbon substrate (Table III).
A graphical representation of the specificity data for wt and mutant
5.2 is presented in Fig. 3.
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Fig. 2.
Plots of initial velocity versus
substrate concentration for castor
9-18:0-ACP desaturase
(A) and mutant 5.2, containing T117R/G188L in wt
background (B). Curves for three substrates are
shown: 18:0-ACP, open circles; 16:0-ACP, solid
circles; and 14:0-ACP, open squares. Each point
represents the mean of three experiments. See Table III for a summary
of kinetic parameters and associated errors.
Kinetic parameters of the wt castor 9-18:0-ACP desaturase
and mutant 5.2
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Fig. 3.
Relationship between log
(kcat/Km) and
chain length of the acyl-ACP substrate for wt castor desaturase
(open circles) and mutant 5.2 (closed
circles). The data are discrete, thus lines are drawn
solely for ease of visualization.
9-position (Fig. 4) showing that the
regiospecificity is unchanged from that of wt desaturase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 desaturases from Milkweed and Doxantha
(5, 11).
9-18:0-ACP desaturase as the
control. The experiments with wt desaturase yielded a
Km value for 18-carbon substrate of 0.46 (±0.05) µM. In contrast, previous studies have reported a range
of Km values: 3.3 (±0.42) µM for (the
same castor desaturase) (7), 0.38 µM for the safflower
enzyme (4), and 13 (±8) µM for a soybean isoform (32).
We have confidence in the values reported here for several reasons.
First, the estimates of substrate concentrations are based on specific
radioactivity of the substrate; second, the values show the smallest
statistical variability; and third, the values are based on a ranges of
substrate concentrations typically from one-fifth to five times the
Km value. All of these conditions were not met in
the studies described above. In addition, the values for wt enzyme
presented here are consistent with the pool sizes of long chain
acyl-ACPs in plants that are in the high nM range (33),
i.e. comparable with the Km for 18:0-ACP, but below the Km for 16:0-ACP.
1 as
compared with 2.8 min
1 for wt enzyme with the
16-carbon substrate. Together, these changes in Km
and kcat resulted in an 82-fold increase in the specificity factor (kcat/Km)
for 16-carbon substrates with mutant 5.2, accompanied by an 18-fold
decrease for 18-carbon substrates (Table III). The decrease in
log(kcat/Km) for wt
desaturase as the substrate length decreases from 18 to 14 carbons
(Fig. 3) is similar to that reported
previously (7). However, the differences in both
KM and kcat values
between wt and mutant 5.2 suggest the factors that contribute to
turnover may be more complex than simply the change in binding energy
as proposed previously for wt enzyme with different substrates (7). Thus for mutant 5.2, the decrease in binding energy of the 16-carbon substrate did not prevent the observed 35-fold improvement in specific
activity.
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Fig. 4.
Gas chromatographic analysis of dimethyl
disulfide-derivitized methyl esters of the reaction product of mutant
5.2 with 16:0-ACP substrate (a) and mass spectrometric
analysis of the product peak (b). The source of the
molecular ion M+ and fragments X and Y are indicated.
In structural terms the change in specificity factor between 18- and 16-carbon substrates likely represents loss of methyl contact at the base of the binding pocket in addition to two methylenes from the wall of the pocket. This has a larger effect on affinity and/or substrate positioning than the loss of interaction of a further two methylenes with the wall of the binding pocket with a 14-carbon substrate. In mutant 5.2, bulkier arginine and leucine residues replace threonine and glycine at the end of the binding pocket, apparently shortening the binding pocket and making it more effective for a 16-carbon substrate but less effective for an 18-carbon substrate. The com2 and com4 enzymes on the other hand are more active on 14- than 16-carbon substrates, presumably reflecting the contributions of other residues to binding affinity or positioning of the substrate for catalysis.
Powerful methods have recently been developed to evolve enzymes in vitro (34). They generally use point mutagenesis and identify improved variants followed by recombination of improved isolates to effect further improvements in the property of interest. A limitation inherent to this approach is that it relies on the relatively small subset of four to seven mutations achievable by single base substitution at each location. We note that neither of the pair of substitutions identified in this study, T117R and G188L, would have been reached by single-base substitution. These results support the contention that saturation mutagenesis can generate superior variants to those arising from the subset of possibilities available via point mutagenesis (35). In this context, amino acids can be regarded as "molecular shims," and the larger the number of shim sizes introduced at a particular amino acid location, the more likely that some "optimal substrate binding geometry" will be approached (36). Performing simultaneous saturation mutagenesis at a number of key locations capable of affecting the property of interest simply extends the number of combinations and permutations of binding site geometry. In this case, the loss of desolvation energy of two methylene groups when binding a 16- rather than an 18-carbon substrate was mostly overcome by improved substrate binding orientation with respect to the active site oxidant and improved substrate fit resulting from occlusion of the floor of the binding pocket.
In summary, these data show that it is possible to alter the
specificity of a desaturase enzyme without substantial degradation of
its kinetic parameters. The 9-16:0-ACP desaturase, 5.2, constructed from a
9-18:0-ACP desaturase is far more
active than either naturally occurring
9-16:0-ACP
desaturases. Taken together, these results suggest it should be
generally possible to reengineer archetypal enzymes with substrate
specificities characteristic of newly evolved enzymes while retaining
the desired stability and/or turnover characteristics of the parental paralog.
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ACKNOWLEDGEMENTS |
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We thank J. Cronan for providing the E. coli MH13 cell line, E. Cahoon for helpful discussion, and M. Bewley for computer graphics assistance.
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FOOTNOTES |
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* This work was supported by the Office of Basic Energy Sciences of the United States Department of Energy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Biology, Bldg. 463, Brookhaven National Laboratory, 50 Bell Ave., Upton, NY 11973. Tel.:
631-344-3414; Fax: 631-344-3407; E-mail: shanklin@bnl.gov.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M102129200
1
Fatty acid nomenclature: X:Y
indicates that the fatty acid contains X numbers of carbon
atoms and Y numbers of double bonds; z indicates that a double bond is positioned
at the zth carbon atom from the carboxyl terminus.
3 All specific activities compared in this report were determined using Anabaena vegetative ferredoxin.
4
Amino acid numbering corresponds to the sequence of the
mature castor 9-18:0-ACP desaturase
(6).
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ABBREVIATIONS |
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The abbreviations used are: ACP, acyl carrier protein; wt, wild-type; PCR, polymerase chain reaction.
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REFERENCES |
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