Jaworski Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3
Trang 1Studies into factors contributing to substrate specificity
of membrane-bound 3-ketoacyl-CoA synthases
Brenda J Blacklock and Jan G Jaworski
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA
We are interested in constructing a model for the
substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase
(FAE1 KCS),the enzyme responsible for production of very
long chain fatty acids of plant seed oils Arabidopsis thaliana
and Brassica napus FAE1 KCS enzymes are highly
homo-logous but the seed oil content of these plants suggests that
their substrate specificities differ with respect to acyl chain
length We used in vivo and in vitro assays of Saccharomyces
cerevisiae-expressed FAE1 KCSs to demonstrate that the
B napusFAE1 KCS enzyme favors longer chain acyl
sub-strates than the A thaliana enzyme Domains/residues
responsible for substrate specificity were investigated by
determining catalytic activity and substrate specificity of
chimeric enzymes of A thaliana and B napus FAE1 KCS
The N-terminal region,excluding the transmembrane domain,was shown to be involved in substrate specificity One chimeric enzyme that included A thaliana sequence from the N terminus to residue 114 and B napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A thaliana FAE1 KCS However,a K92R substitution in this chimeric enzyme changed the specificity to that of the B napus enzyme without loss of catalytic activity Thus,this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity
Keywords: Arabidopsis thaliana; Brassica napus; fatty acid elongation; 3-ketoacyl-CoA synthase
The very long chain fatty acids (VLCFA) found in seed
oils are derived from the elongation of products of de novo
fatty acid biosynthesis [1] The initial reaction of
elonga-tion,i.e the iterative condensation of acyl units with
malonyl-CoA,is catalyzed in the seed by the
membrane-bound fatty acid elongase-1 3-ketoacyl-CoA synthase
(FAE1 KCS) [2] Subsequent reduction and dehydration
reactions are carried out by distinct and separate enzymes
that are just beginning to be characterized [1,3,4]
FAE1 KCS was first identified in Arabidopsis thaliana [5]
and homologues have been found in oleaginous species
such as Brassica napus, B juncea,and Simmondsia
chinen-sis[6–10] The functional similarity among these enzymes
is demonstrated by the ability of the jojoba FAE1 KCS to
complement the canola fatty acid elongation mutation
even though jojoba produces wax rather than
triacylgly-cerol,as found in other seed oils [6]
Examination of the VLCFA content of the seed oils of
A thalianaand B napus reveals differences in the levels of
eicosenoic (20:1) and erucic (22:1) fatty acids In A thaliana
seed oil,20% of the total fatty acids are VLCFA of which
18% of the total fatty acids are in the form of 20:1 and 2%
in the form of 22:1 [11] In B napus seed oil,62% of the total fatty acids are monounsaturated VLCFA,10% as 20:1 and 52% as 22:1 [12] As FAE1 KCS is responsible for VLCFA production in oilseeds [2,5,6], this diversity in VLCFA content suggests that A thaliana and B napus FAE1 KCS enzymes have distinct substrate specificities with the B napus enzyme favoring longer chain length substrates than the A thaliana enzyme The high sequence identity (86%) between these two enzymes further suggests that the determinants responsible for fatty acid substrate specificity in FAE1 KCS are few and potentially identifi-able
The significant amino acid sequence homology of FAE1 KCSs with soluble condensing enzymes,such as chalcone synthase and 3-ketoacyl-acyl carrier protein synthases (KASs) is consistent with a role for FAE1 KCSs
as fatty acid condensing enzymes [5,6,13] Our understand-ing of the structure/function relationships of soluble condensing enzymes has been greatly advanced with the recent crystal structures of KAS I,-II,and -III and chalcone synthase [14–19] However,only limited information is available about the structure of the membrane-bound KCSs Secondary structural analysis of the family of FAE1 KCS enzymes reveals two putative transmembrane domains at the N termini of the proteins Recent work in our laboratory has confirmed that the amino terminus of Arabidopsis FAE1 KCS is involved in anchoring the enzyme to the membrane [20] The difficulty inherent in crystallizing membrane-bound enzymes required us to take
a different approach to probing the structure/function relationships of FAE1 KCS Here,we report utilization of a domain-swapping approach to investigate the structural domains and residues responsible for substrate specificity in FAE1 KCS
Correspondence to: B J Blacklock,Department of Chemistry and
Biochemistry,Miami University,Oxford,OH 45056,USA.
Fax: +1 513 529 5715,Tel.: +1 513 529 1641,
E-mail: blacklb@muohio.edu
Abbreviations: VLCFA,very long chain fatty acid; FAE1 KCS,fatty
acid elongase-1 3-ketoacyl CoA synthase; KAS,3-ketoacyl-acyl
carrier protein synthase; cm-ura,complete minimal dropout media
lacking uracil; FAME,fatty acid methyl ester.
Note: The SWISS-PROT accession numbers for the FAE1 KCS are:
Arabidopsis thaliana,Q38860; Brassica napus,O23738.
(Received 31 May 2002,revised 2 August 2002,
accepted 12 August 2002)
Trang 2The redesign of a number of plant lipid metabolic
enzymes by swapping domains between related yet
func-tionally divergent enzymes has proven useful in obtaining
catalysts with novel substrate specificity An understanding
of the regions of enzymes that contribute to substrate
specificity and catalytic activity has also been gleaned from
these studies [21–25] By replacing only five amino acid
residues,Cahoon and coworkers engineered a soluble fatty
acid desaturase with D6-16:0 substrate specificity to one that
functions principally as a D9-18:0 desaturase [21] Similarly,
specificity for a 16:0 substrate was imparted to a D9-18:0
desaturase by replacement of a single residue [22]
An analogous approach was taken to deciphering the
architecture of the substrate-binding site of
membrane-bound desaturases and related enzymes [24,26] Libisch
et al constructed chimeras of Borago officinalis D6-fatty
acid and D8-sphingolipid desaturases and analyzed effects
on substrate specificity [24] These studies were unsuccessful
in identifying a discrete domain that can differentiate
between a phospholipid-conjugated substrate and a
cera-mide-conjugated substrate However,the authors were
successful in modifying the substrate specificity of the
D6-fatty acid desaturase to a preference for shorter chain
fatty acids [24]
A similar study demonstrated a switch in the oxidative
reactions catalyzed by membrane-bound oleate desaturases
and hydroxylases upon site-directed mutagenesis [26] When
the amino acid sequences of these highly homologous
enzymes were compared,seven residues were identified that
were conserved in the desaturases but divergent in the
hydroxylases The mutation of these residues in the
desaturases to the corresponding hydroxylase residues
resulted in the conversion of desaturase activity to
hydroxy-lase activity The reciprocal experiment allowed the
conver-sion of a hydroxylase to a desaturase [26]
The objective of our work was to examine the substrate
specificity of A thaliana and B napus FAE1 KCSs and to
study the determinants of fatty acyl chain length specificity
in 3-ketoacyl CoA condensing enzymes We were able to
map residues and regions of primary structure involved in
substrate specificity in KCS enzymes These studies
repre-sent the first steps toward a characterization of the
substrate-binding site of the membrane-bound KCS enzymes
E X P E R I M E N T A L P R O C E D U R E S
Construction of yeast expression vectors and cell lines
A thaliana (var WS) FAE1 and B napus (var Golden)
FAE1.1genes were amplified from plasmids containing the
cDNAs with Vent DNA polymerase (New England
Biolabs) using primers (Table 1) containing restriction sites
convenient for subcloning into the pYES2 expression vector
(Invitrogen) PCR products and DNA fragments were
purified by agarose gel electrophoresis followed by Gene
Clean (Bio101,Vista,CA) A thaliana, B napus and
At114K92R genes were subcloned into a pYES2 vector
engineered to encode a (His)6GlySer fusion protein through
BamHI/EcoRI restriction enzyme sites Purified,digested
insert and pYES2 vector were ligated with T4 DNA ligase
(Life Technologies Gibco BRL) and transformed into
competent XL-1 Blue Escherichia coli (Stratagene) by
standard techniques [27] Insert-containing plasmids were
sequenced by in-house automated DNA sequencing to ensure they were mutation-free Plasmids were transformed into competent Saccharomyces cerevisiae (strain InvSc1, Invitrogen) by the lithium acetate method of Geitz et al [28]
Domain swapping and site-directed mutagenesis
by overlap extension Chimeric genes were constructed either by restriction enzyme digestion when convenient sites for domain swap-ping were present,or by thermostable DNA polymerase-mediated overlap extension and PCR essentially as described [29,30] Briefly, gene fragments were amplified from plasmids containing A thaliana or B napus FAE1 using 5¢ or 3¢ specific primers with restriction sites and overlapping primers either straddling the desired splice site
of the chimeric gene or containing the desired site-directed mutation (Table 1) Purified fragments were mixed and the full-length chimeric gene was constructed by extension of the overlapping fragments and PCR amplification with extreme 5¢ and 3¢ primers in the same reaction Amplifica-tion products were subcloned into pYES2 and sequenced as described above to confirm the correct sequence
Expression of FAE1 KCSs in yeast and preparation
of microsomes Transformed yeast was grown overnight with shaking in rich media at 30C and was used to inoculate complete minimal dropout media lacking uracil (cm-ura) [31] supple-mented with 2% galactose to give an initial D600of 0.01– 0.04 The cm-ura + gal cultures were grown to 1.5–2 D600 units and harvested Microsomes were prepared as des-cribed previously [32] using ice-cold isolation buffer (80 mM
Hepes/KOH pH 7.2,5 mMEGTA,5 mMEDTA,10 mM
KCl,320 mM sucrose,2 mM dithiothreitol) Protein con-centrations were determined after the method of Bradford [33] using BSA as a standard and adjusted to 2.5 lgÆlL)1by the addition of glycerol to 15% and isolation buffer Microsomes were frozen on dry ice,stored at)80 C until use and,once thawed,were not refrozen
GC/MS analysis of yeast lipids Yeast transformed with pYES2 or pYES2 with FAE1 insert were grown in cm-ura plus 2% raffinose and were induced
by 2% galactose as described above Cells were harvested by centrifugation and washed with dH2O; methyl esters of cellular lipids were prepared by incubation of the cell pellet
in 2% H2SO4in methanol at 80C for 1–2 h Fatty acid methyl esters (FAMEs) were extracted into hexanes, concentrated by evaporation under an N2 stream and dissolved in a small volume of hexanes FAMEs were separated by GC (Thermoquest Trace GC) on an RTX-5MS 0.25 lm column (Restek Corp Bellefonte,PA) and identified by MS on an inline Finnigan Polaris mass spectrometer
Assay of elongation activity of FAE1 KCSs Fatty acid elongase activity was measured essentially as described previously [34] The elongation reaction consisted
Trang 3of 20 mM Hepes/KOH pH 7.2,20 mM MgCl2,500 lM
NADPH,10 lM CoASH,100 lM malonylCoA,15 lM
[1-14C]18:1CoA,and 6 lg protein of prepared microsomes
in a final volume of 25 lL [1-14C]18:1CoA was synthesized
from [1-14C]18:1 free fatty acid (50–55 lCiÆlmol)1; ICN,
Costa Mesa CA) as described by Taylor et al [35] Methyl
esters of the radiolabeled acyl CoA elongation products
were prepared as described above,separated by reversed
phase silica gel TLC (Alltech,Deerfield,IL) with
acetonit-rile/methanol/H2O (65 : 35 : 0.5,v/v) developing solvent
[36] and analyzed by phosphorimaging; each band was then
quantitated by ImageQuant software (Molecular
Dynam-ics,Inc.)
Solubilization and isolation of (His)6-tagged fusion
proteins
(His)6 fusion proteins were solubilized and isolated as
described [20] Microsomes (2 lg proteinÆlL)1) expressing
(His)6-tagged fusion proteins were solubilized by incubation
on ice for 2 h in solubilization buffer (320 mMNaCl,0.5%
Triton X-100) and insoluble material was pelleted by
ultracentrifugation at 4C for 1 h at 235 000 g Superna-tants were combined with binding buffer (25 mMsodium phosphate pH 8.0,0.5% Triton X-100,150 mMNaCl,10% glycerol) at a ratio of 0.43 : 1 (supernatant : binding buffer) and applied to a 300-lL column of Ni+2-pentadentate chelator (Ni-PDC,Affiland,Ans-Liege,Belgium) The column was washed with 1 mL binding buffer,1 mL wash buffer (25 mM sodium phosphate pH 8.0,0.5% Triton X-100,500 mMNaCl,10% glycerol,20 mMimidazole) and
1 mL binding buffer Proteins were eluted from the column with 300 lL elution buffer (25 mM sodium phosphate
pH 8.0,0.5% Triton X-100,150 mMNaCl,10% glycerol,
300 mMimidazole),dithiothreitol was added to 2 mM,and samples were frozen on dry ice and stored at)80 C Condensation assay of (His)6-FAE1 KCS
Condensation activity of (His)6-FAE1 KCSs was assayed as previously described [37] Briefly,2.5 lL purified sample was incubated with condensation reaction mix (10 mM
sodium phosphate,pH 7.2,0.05% Triton X-100,15 lM
acyl CoA,20 l [3-14C]malonyl CoA) where the acyl CoA
Table 1 Primers used in subcloning and overlap PCR.
Trang 4was either 18:1CoA or 20:1CoA for 10 min at 30C.
Radiolabeled malonyl CoA was prepared as described
previously [38] 3-ketoacyl CoA reaction products were
reduced to the diols with NaBH4,extracted into petroleum
ether,concentrated by evaporation under an N2stream,and
quantified by liquid scintillation analysis
R E S U L T S
This report describes studies of the fatty acid chain length
substrate specificity of FAE1 KCS We tested the
hypothe-sis that A thaliana and B napus FAE1 KCS enzymes have
divergent substrate specificity and that determinants of that
specificity can be identified by domain swapping
experi-ments
We have developed a facile biochemical method to
characterize fatty acid elongase activities using an S
cere-visiaeexpression system Endogenous yeast fatty acids serve
as elongation substrates for in vivo analysis [2] and
exogenously supplied radiolabeled 18:1CoA is readily taken
up by microsomal fractions for in vitro assays [39]
As a first step,we established that the A thaliana and
B napus FAE1 KCSs were active in the S cerevisiae
expression system and indeed had distinct substrate
speci-ficities Elongation of yeast endogenous fatty acids by
FAE1 KCSs was examined by expressing plant
FAE1 KCSs in yeast and then analyzing the fatty acid
content of cellular lipids Table 2 shows the C20 to C26
fatty acid content of cellular lipids when yeast transformed
with pYES2 with A thaliana or B napus FAE1 inserts or
the empty vector was grown under galactose induction
conditions The VLCFA content of yeast carrying the
empty vector was principally limited to 26:0 with little or no
C20,C22 or C24 fatty acids present In contrast,expression
of either A thaliana or B napus FAE1 KCS resulted in the
production of significant levels of both saturated and
unsaturated C20 to C26 VLCFA A thaliana FAE1 KCS
expression caused an increase in VLCFA methyl esters to
32% of the total FAMEs: 19.3% C20,5.8% C22,2.6% C24
and 4.3% C26 B napus FAE1 KCS expression,on the
other hand,produced 11% of the total FAMEs as VLCFA:
3.5% C20,2.1% C22,0.8% C24 and 4.1% C26 These data
demonstrate that plant FAE1 KCS enzymes were expressed
and have activity in this yeast system Further,these results
show that in yeast, A thaliana FAE1 KCS produces more
C20,C22 and C24 fatty acids than B napus FAE1 KCS
and that the majority of the VLCFA products are C20
B napus FAE1 KCS appeared to have more activity
toward C20 than A thaliana FAE1 KCS as indicated by
near equal levels of C20 and C22 products when B napus FAE1 KCS was expressed While these results are,in general,consistent with the apparent substrate specificity of the A thaliana and B napus FAE1 KCS enzymes in seeds, the B napus enzyme appeared to be less active than the
A thalianaFAE1 KCS in yeast
The substantial levels of saturated and unsaturated fatty acid methyl esters demonstrated that FAE1 KCSs have activity toward both monounsaturated and saturated fatty acids The FAE1 KCS-derived saturated fatty acid prod-ucts appear to feed into the yeast saturated VLCFA pathway VLCFA up to C26 as 24:0 and 26:0 were produced at elevated levels when either A thaliana or
B napus FAE1 KCS were expressed compared to the empty vector control
While in vivo assays indicated that the plant FAE1 KCSs were able to couple to the yeast fatty acid elongation complex,an assay of the actual catalytic activity of the FAE1 KCSs was required to characterize the enzymes accurately Microsomes were prepared from galactose-induced yeast transformed with empty vector (pYES2) or pYES2 with A thaliana or B napus FAE1 insert Elonga-tion of [1-14C]18:1CoA to [3-14C]20:1CoA and [5-14C]22:1CoA by the microsomes was measured by incubation with malonyl CoA and required cofactors, followed by transacylation of the CoA conjugates to methyl esters [34,39] Elongation activity of both A thaliana and
B napusFAE1 KCS reached a plateau by 20 min but the catalytic activity of B napus FAE1 KCS was lower than that of A thaliana FAE1 KCS (Fig 1) When the activities
of the two enzymes were compared in numerous experi-ments with individual transformants,this difference between A thaliana and B napus FAE1 KCSs was consis-tently observed As the same expression vector and yeast strain were used,this appears to reflect actual differences in catalytic activity rather than differences in expression level although we do not know the amount of FAE1 KCS protein present in the microsomes It appears that the
A thaliana FAE1allele codes for a more active enzyme than the B napus FAE1 allele used here Subsequently,we were able to isolate (His)6-tagged fusion proteins and directly assay condensation activity of A thaliana and B napus (His)6-FAE1 KCSs [A thaliana,538.5 ± 68.1 pmolÆlg)1 protein (± SD) 20:1 product and 126.8 ± 14.7 22:1 product;
B napus 135.0 ± 17.5 20:1 product and 64 ± 4.4 22:1 product] These observations are consistent with results obtained in in vivo experiments
Comparison of total elongation activities demonstrated that the A thaliana enzyme was more active than the
Table 2 Effect of the expression of A thaliana and B napus FAE1 KCS on yeast cellular lipids S cerevisiae transformed with expression vectors encoding A thaliana or B napus FAE1 KCS or the empty vector (pYES2) were pre-grown on raffinose (2%) and induced with galactose (2%) for
2 days Total fatty acid composition was determined by GC/MS of FAMEs prepared by transacylation Results are reported as the percentage total fatty acid methyl esters and are the mean ± SD of five independent transformants.
FAME (%)
Trang 5B napusenzyme (Table 3) Analysis of the individual acyl
products indicated differences in the substrate specificity
of the two FAE1 KCSs Relative substrate specificity of
FAE1 KCSs can be compared by expressing the ratio of
22:1 produced over 20:1 produced An enzyme which favors
the production of 22:1 would therefore have a higher 22:1/
20:1 product ratio than one which favors the production of
20:1 fatty acids Table 3 shows the product ratios of
A thaliana and B napus FAE1 KCSs after 10 min The
larger 22:1/20:1 product ratio of B napus FAE1 KCS
demonstrated that B napus FAE1 KCS was indeed
pro-portionally more active toward 20:1 as a substrate than the
A thaliana FAE1 KCS A thaliana FAE1 KCS had a
substrate specificity directed primarily toward an 18:1
substrate
Both in vivo and in vitro data indicate that A thaliana
and B napus FAE1 KCS enzymes have distinct substrate
specificities and that differences in seed oil composition
reflect these substrate specificities The primary sequence
alignment (Fig 2) demonstrates that there is a high degree
of homology between A thaliana and B napus
FAE1 KCS (86% identity) This similarity suggested to
us that factors involved in substrate specificity were
potentially identifiable As little is known about the
structure of FAE1 KCSs,we approached the problem
with no attempt to predict where residues or regions
important in substrate specificity would be found
Swap-ping of domains between A thaliana and B napus
FAE1 KCSs by ligation of restriction digest fragments or
by overlap PCR produced cognate full-length chimeric FAE1 genes Fig 3 is a schematic representation of a number of the chimeric enzymes engineered in this study Chimeric enzymes were characterized based on activity and the 22:1/20:1 product ratio Our goal was to identify the smallest domain required to retain a particular substrate specificity and to engineer an enzyme with novel activity Twenty-nine chimeras were prepared for this study The first group of constructs produced pairs of chimeras with switchover points between A thaliana FAE1 KCS sequence and B napus FAE1 KCS sequence at residues 399,254 and 173 These constructs represent swaps in the C-terminal domain,at the midpoint and in the N-terminal domain (Fig 3) Chimeras were named based on the origin
of the N terminus and the residue at which the switchover occurred For example,for At254,residues 1–254 are derived from A thaliana FAE1 KCS and residues 255–506 are derived from B napus FAE1 KCS When in vitro elongation activity was measured,the initial FAE1 KCS chimeras segregated into two classes: those with measurable elongase activity and those with little or no activity (Fig 4A) All of the chimeric enzymes with activity had
A thalianasequence at the N terminus while enzymes with
B napuscoding sequence in the N terminus had little or no activity All of the active chimeras had A thaliana-like substrate specificity as indicated by low 22:1/20:1 product ratios (Fig 4B) The FAE1 KCS chimera with the smallest region containing A thaliana FAE1 KCS sequence in this group was At173 indicating that at least one determinant of
Table 3 Elongation activity and product ratio (22:1/20:1) for A thaliana and B napus FAE1 KCS Reactions were carried out for 10 min and results presented are the mean ± SD of five individual assays using four separate microsomal preparations.
16
14
12
10
8
6
4
2
0
50 40
30 20
10
0
time (min)
Fig 1 Elongase activity of microsomes prepared from S cerevisiae
expressing A thaliana or B napus FAE1 KCS Microsomes prepared
from induced S cerevisiae transformed with empty vector (pYES2, n)
or vectors encoding A thaliana (h) or B napus (s) FAE1 KCSs were
were taken between 5 and 45 min and are plotted as total elongation
results are the mean of five assays using four separate microsomal
preparations and error bars indicate standard deviation.
FAE1 KCS sequences Identical residues are included in the shaded box.
Trang 6fatty acyl chain length specificity in FAE1 KCSs resides in
the N terminal one-third of the protein
We further dissected the N-terminal region of the
FAE1 KCSs by preparing chimeras with switchover
points at residues 114 and 74 (At114 and At74) Although
the catalytic activity was lower in these chimeras,At114
had A thaliana FAE1 KCS-like substrate specificity while
At74 more closely resembled B napus FAE1 KCS in
substrate specificity (Table 4) At74 had primarily
B napussequence (85%) with A thaliana sequence
inclu-ded only in the extreme N-terminal putative
transmem-brane domains Thus,a shift in substrate specificity from
A thaliana FAE1 KCS-like to B napus FAE1 KCS-like
occurred between chimera At114 and At74 indicating that
determinants of substrate specificity reside between
resi-dues 74 and 114 Furthermore,as At74 is encoded entirely
by B napus sequence except for the transmembrane
domain,the transmembrane domain appears to have
little or no role in the determination of substrate
specificity in FAE1 KCS
The stretch of primary sequence between residues 74 and
114 contains nine nonidentical residues (Fig 2)
Site-direc-ted mutagenesis of chimera At114 was utilized to dissect this
region further in an attempt to reveal specific residues
involved in imparting specificity toward the 20:1 substrate in
FAE1 KCSs Some of these chimeras were inactive
(At114D81E,At114P89T,At114L91C,At114V93S),while
others had activity similar to that of the parent enzymes
(Fig 5A) When the 22:1/20:1 product ratio of the active
chimeras were compared,all chimeras in this group had
A thaliana-like product ratios except At114K92R,which
had the substrate specificity of B napus FAE1 KCS (Fig 5B) This suggests that residue 92 has some role in determining substrate specificity of FAE1 KCSs Catalytic activity of At114K92R,however,was similar to that of
A thalianaFAE1 KCS (Fig 5A) while At114 had lower catalytic activity than A thaliana FAE1 KCS This dem-onstrated a novel activity in At114K92R and suggested that residue 92 is involved in catalytic activity as well as substrate specificity
We further examined the role that residue 92 plays in substrate specificity by replacing K92 with R in wild-type
A thaliana FAE1 KCS Both catalytic activity and
25
20
15
10
5
0
A thaliana B napus
At399 Bn399 At254 Bn254 At173 Bn173
A
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A thaliana B napus
At399 At254 At173
B
A thaliana, B napus, At399, Bn399, At254, Bn254, At173, and Bn173 FAE1 KCS and (B) product ratio (22:1/20:1) for A thaliana, B napus, At399, At254, and At173 FAE1 KCS (A) Reactions were carried out for 10 min and results presented are the mean of one to three indi-vidual assays using three microsomal preparations (± SD) (B) Reactions were carried out for 10 min and results presented are the mean of one to three individual assays using three microsomal preparations (± SD).
Fig 3 Schematic representation of chimeric FAE1 KCS polypeptides.
and B napus FAE1 KCS sequence is represented as a shaded bar.
The chimeric alleles were named for the FAE1 KCS sequence in
the N-terminal domain followed by the residue number at which the
sequence shifts to the other FAE1 KCS sequence Point mutations are
named by the convention of the wild-type residue followed by the
residue number and the amino acid that has been substituted at that
residue.
Table 4 Elongation activity and product ratio (22:1/20:1) for A thali-ana, B napus, At114 and At74 F AE1 KCS Reactions were carried out for 10 min and results presented are the mean ± SD of two or three individual assays for each of four microsomal preparations.
Trang 7substrate specificity of AtK92R were essentially identical to
the wild-type A thaliana FAE1 KCS (Fig 5)
A procedure for the isolation of (His)6-tagged
FAE1 KCSs was developed in our laboratory,subsequent
to the analysis of the entire set of chimeras prepared in this
study,which allowed us to assay directly the condensation
activity of the isolated (His)6-FAE1 KCSs [20] We used this
assay to verify that the product ratios observed in the
elongation assay of microsomal preparations were an actual
measure of the activity of the FAE1 KCS and were not an
artifact of differential interactions with yeast enzymes
required to produce the elongation product When the
condensation activity of isolated (His)6-tagged A thaliana,
B napus,and At114K92R FAE1 KCSs were measured
with 18:1CoA and 20:1CoA substrates,22:1/20:1 product
ratios were: (His)6-A thaliana FAE1 KCS,0.25 ± 0.07;
(His)6-B napus FAE1 KCS,0.78 ± 0.13; (His)6
-At114K92R,0.71 ± 0.10 (product ratio ± SD,n¼ 9)
The product ratio of condensation activity of (His)6
-B napusFAE1 KCS and (His)6-At114K92R was therefore
similar and distinct from that of (His)6-A thaliana
FAE1 KCS as was demonstrated by the elongation assay
of FAE1 KCSs in the yeast microsomal system
D I S C U S S I O N
In this study,we attempted to gain insights into the structural basis for substrate specificity in FAE1 KCS enzymes As no crystal structure of any membrane-bound FAE1 KCS is available,we used a biochemical approach with molecular genetic tools We reasoned that the difference in oil content of A thaliana and B napus seeds is a consequence of divergent substrate specificity of the key enzyme responsible for VLCFA production in seeds,FAE1 KCS The high degree of homology between these enzymes allowed us to use a domain-swapping approach to identify regions/residues involved in substrate specificity
Our first step was to establish the substrate specificity of
A thalianaand B napus FAE1 KCSs with both in vivo and
in vitroassays of elongation activity Analysis of the effect of expression of A thaliana and B napus FAE1 KCS on yeast cellular lipids indicated that these enzymes have activity toward both monounsaturated and saturated fatty acids This is consistent with the observation by Millar and Kunst that over-expression of FAE1 KCS in Arabidopsis resulted
in an increase in elongation products of both 18:0 and 18:1 fatty acids [2] In addition,James et al showed that disruption of expression of FAE1 KCS in Arabidopsis resulted in a decrease in the levels of both saturated and monounsaturated VLCFA in seed [5] In the in vivo experiment presented here,C18 and C20 monounsaturated fatty acids were elongated by the FAE1 KCSs to a greater degree than were saturated fatty acids This may reflect the available substrates in this yeast system and/or the ability of the enzymes further down the metabolic pathway to use the products of the FAE1 KCS condensation activity Indeed,
in the S cerevisiae strain used in these studies,monoun-saturated C16 and C18 fatty acids were at least twice as abundant as the saturated C16 and C18 fatty acids The in vivo activities presented here demonstrated that both FAE1 KCS enzymes were active in the yeast expres-sion system and were able to couple to the yeast fatty acid elongation complex These results show the usefulness of this in vivo system as a quick screen for activities of FAE1 KCSs However,the results are reflective of the entire yeast metabolic pathways from the synthesis of 3-ketoacyl CoAs by a condensing enzyme to the incorporation of fatty acids into lipids such as triacylglycerol or membrane phospholipids This in vivo system is therefore inadequate for assigning substrate specificity and catalytic activity to an exogenous KCS as activities and specificities of subsequent yeast enzymes,potential compartmentalization and avail-ability of substrates could interfere with interpretation of the results This may be especially important in examining the activity of other plant KCS enzymes such as those encoded
by KCS1 and CER6 which use longer VLCFA as substrates [39,40] A detailed examination of activity and substrate specificity of a KCS is possible only with an in vitro approach
We found that in both in vivo and in vitro assays,
A thaliana FAE1 KCS had more activity toward a C18 substrate than toward a C20 substrate The B napus enzyme,on the other hand,had similar activity toward C18 and C20 fatty acyl groups The data presented here demonstrate that A thaliana and B napus FAE1 KCS enzymes have distinct substrate specificities
20
15
10
5
0
0.5
0.4
0.3
0.2
0.1
0.0
B
A
At114K92R, At114V93S, At114V95I, At114I105 V, At114T110P,
At114S112-, AtK92R FAE1 KCS and (B) product ratio (22:1/20:1) for
At114T110P, At114S112-, AtK92R FAE1 KCS (A) Reactions were
carried out for 10 min and results presented are the mean of one to
three individual assays each of at least seven separate microsomal
preparations (± SD) (B) Reactions were carried out for 10 min and
results presented are the mean of one to three individual assays each of
at least seven separate microsomal preparations (± SD).
Trang 8and are consistent with our hypothesis that differences in
seed oil composition reflect these substrate specificities
Through the preparation and assay of chimeric enzymes,
we were able to define the region between residue 74 and 173
as an important domain in conferring substrate specificity to
FAE1 KCSs This domain excludes the putative
transmem-brane domain predicted to be to the N-terminal side of
residue 72 It is not surprising that the transmembrane
domain of FAE1 KCSs is not involved in substrate
specificity and therefore substrate binding because the fatty
acyl CoA substrates are expected to be delivered from the
cytosol and available at the membrane interface [1] This is
in contrast to membrane-bound fatty acid- and
sphingo-lipid-desaturases,which appear to utilize phospholipid- or
sphingolipid-conjugated fatty acids Chimerigenesis studies
of the borage D6-fatty acid and D8-sphingolipid desaturases
indicate that a transmembrane portion of these enzymes is
involved in substrate binding which is consistent with the
membrane localization of the substrates of these desaturases
[24]
If the region from the end of the transmembrane domain
to residue 173 is indeed involved in imparting substrate
specificity to KCS condensing enzymes,we would expect
this region to be of particular heterogeneity among the
family of plant ketoacyl-CoA condensing enzymes The
completion of the sequence of the Arabidopsis genome [41]
presents an opportunity to compare the sequences of all
predicted KCS-like genes in the genome A recent database
search revealed at least 15 distinct KCS-like genes in the
Arabidopsisgenome The gene product of four of these have
been assigned physiological functions in mutant analysis
and expression disruption studies; FAE1,CER6,KCS1,
and FDH [2,5,39,40,42,43] Alignment of these sequences
demonstrated regions of high homology distributed
throughout the enzymes The region corresponding to
residues 74–173 of A thaliana FAE1 KCS,however,is
relatively heterogeneous Recent work in our laboratory has
identified the active site cysteine of A thaliana FAE1 KCS
as residue 223 and has suggested that His391 is also involved
in the active site [44] These two catalytically important
residues are found in regions of high homology and are
distant from the region that has been identified to be
important in substrate specificity in this study
Given the similarity of the two interchanged residues,it is
surprising that the change of K92 to R in At114 resulted in a
shift of substrate specificity from A thaliana-like to one
more specific for a 20:1 substrate When the substitution of
amino acids in families of proteins such as globins and
cytochrome c was calculated,arginine and lysine were often
substituted for each other and were considered safe
substitutions [45] The absence of an effect of the K92R
mutation in wild-type A thaliana FAE1 KCS points to a
role for residue 92 in determining substrate specificity that is
specific to At114 FAE1 KCS Arg92 in At114K92R
appears to interact with B napus residues that are not
present in A thaliana FAE1 KCS These results also
indicate that residue 92 is not the sole residue involved in
conferring substrate specificity in FAE1 KCS The chimera
At114K92R will therefore be invaluable in further studies of
the domains/residues that are involved in substrate
speci-ficity in FAE1 KCS as this enzyme may now be utilized in
domain-swapping experiments that focus on the C termini
of the proteins
We noted the levels of enzyme activity of A thaliana and
B napusFAE1 KCSs with interest B napus seed oil has a larger proportion of VLCFA compared to total fatty acids than A thaliana seed oil The incongruity of the level of seed oil VLCFA and the activity of the B napus FAE1 KCS assayed here may have several explanations A less active FAE1 KCS in B napus seeds could be compensated by the efficiency of the remainder of the biosynthetic pathway At least five very similar genes encoding FAE1 KCS enzymes have been found in B napus cultivars [7,8,46,47] The fatty acid content of the seed oils of the cultivars from which these genes were cloned may reflect differences in elongation activity The FAE1 KCS from B napus (var Golden) used
in this study may have a relatively low level of activity compared to other B napus FAE1 KCS homologues In addition,the amphidiploidy of Brassica species such as
B napus[12] may result in a higher seed oil VLCFA level than that predicted from the in vitro activities of individual FAE1 KCSs In the plant,one highly active FAE1 KCS may dominate or the additive effect of expression of two different FAE1 KCSs may result in high VLCFA production
In this work we also noted the level of activity found in many of the chimeric enzymes Many of the changes we made resulted in chimeric enzymes with substantially lower activity than the wild-type parents This was especially apparent when the N terminus of the chimeras was derived from B napus FAE1 KCS In addition, chimeras At74 and At114 had relatively poor activity compared to the wild-type enzymes When similar experi-ments were conducted with plant acyl-acyl carrier protein thioesterases and acyl-acyl carrier protein desaturases,a decrease in the activity of the engineered enzymes often resulted,regardless of a high level of identity between the two enzymes examined [21–23] On the other hand, several of the changes made in our study resulted in enzymes with excellent retention of activity For example, At114K92R had even more activity than the At114 parent chimera
The crystal structures of related condensing enzymes,
E coliKAS I,-II,and -III,alfalfa chalcone synthase and
S cerevisiae3-ketoacyl-CoA thiolase,have been solved [15– 19,48] Although the overall sequence homology is very low, these enzymes exhibit a common fold with a five-layered core structure; a-b-a-b-a,where a comprises two a-helices and each b is a five-stranded,mixed b-sheet [16,18,19] Alignment of the secondary structural features of the crystal structures with PHD predicted secondary structure of FAE1 KCS (data not shown) suggesting that there are common structural elements among these condensing enzymes The substrate-binding site of KAS II may repre-sent the best available model for that of FAE1 KCS,as KAS II catalyzes the condensation of a 16:1 moiety with malonyl-ACP The crystal structure of KAS II alone and with the mycotoxin inhibitor,cerulenin,revealed that the substrate binding pocket of KAS II is lined with hydro-phobic residues predominantly from the N-terminal domain
of the enzyme that are essential for the binding of long chain substrates such as 16:1 [16,49] The research presented here demonstrates that residues important to substrate binding for FAE1 KCS also include N-terminal residues and may suggest similar substrate-binding pockets for the two enymes
Trang 9In the redesign of a soluble fatty acid desaturase from one
with D6-16:0 activity to one with D9-18:0 activity,Cahoon
et al replaced only five amino acids in the D6-16:0 specific
enzyme with corresponding residues from the D9-18:0
specific desaturase [21] The X-ray crystal structure of the
D9-18:0 desaturase revealed that many of the residues which
were identified in the mutagenesis study to be responsible
for chain length substrate specificity,line the
substrate-binding pocket [21,50] The substitution of only two residues
is sufficient for the conversion of an 18:0-specific desaturase
to one that strongly prefers a 16:0 substrate albeit with less
than half of the total catalytic activity of the parent enzyme
[21] Taken together with our studies,these results suggest
that the substrate-binding site of the soluble desaturases is
more rigid in the nature of acceptable substrates than that of
FAE1 KCS The KCS enzymes studied here appear to have
a flexible substrate-binding pocket as demonstrated by the
in vivoactivities in yeast The conversion of an 18:1-specific
condensing enzyme to one that uses only 20:1 substrates
may not therefore be feasible with the replacement of a
small number of residues
In summary,this work was successful in identifying,for
the first time,regions and residues important in fatty acyl
chain length specificity in a membrane-bound condensing
enzyme The identification of At114K92R as an enzyme
with novel activity will be useful for the future identification
of other residues involved in fatty acyl chain length substrate
specificity in FAE1 KCS This study will also serve as a basis
for progress toward further understanding the structure–
function relationships for FAE1 KCS and other
membrane-bound fatty acid elongase condensing enzymes
A C K N O W L E D G E M E N T S
We thank Dr Hugo Dooner,Rutgers University,for permission to use
the A thaliana FAE1 gene and Dr Ljerka Kunst,University of British
Columbia,for the generous gift of the B napus FAE1 cDNA This
work was supported by a grant from Cargill,Inc.
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