(Received for publication, October 25, 1995; and in revised form, November 16, 1995)
From the
The plant acyl-acyl carrier protein (acyl-ACP) thioesterases
(TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of
their utilities in the genetic engineering of plant seed oils.
Biochemical data have shown the possible involvement of an active-site
cysteine and a histidine in catalysis, suggesting that these enzymes
activate the hydrolysis of the thioester bond using the same basic
catalytic machinery as those of proteases and lipases. To identify the
cysteine and histidine residues that are critical in catalysis we
substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine
(Cys-320) to an Ala or a Ser, and three conserved histidines (His-140,
His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a
substantial loss of enzyme activity. However, only C320A and H285A
completely inactivated the enzyme, indicating that these two residues
are essential for catalysis. Considerable activity (>60%) still
remained when Cys-320 was converted to a Ser, but this mutant (C320S)
displayed a reversed sensitivity toward thiol or serine hydroxyl
inhibitors compared with the wild-type enzyme. A pH optimal study
demonstrates that while the wild-type enzyme has the highest activity
between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to
higher pH and a significant increase of activity around pH 12. This
result suggests that Arg-285 (pK 12) is
deprotonated at high pH, thus partially mimicking the role of His-285
for proton abstraction in the wild-type enzyme. We conclude that the
Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can
attack the thioester bond of the substrate 12:0 ACP, assisted by
His-285. Because plant TEs are highly conserved in length and sequence
and the residues investigated here are completely conserved in all
available TEs, it is reasonable to believe that homologues of Cys-320
and His-285 are present in the active sites of all plant acyl-ACP TEs.
A plant thioesterase (TE) ()removes the acyl moiety
from the acyl-acyl carrier protein (ACP), releasing it as a free fatty
acid. The plant acyl-ACP TEs play an essential role in chain
termination during de novo fatty acid synthesis and have been
proven to be important in plant fatty acid bioengineering(1) .
In higher plants, these enzymes can be classified into two distinct but
related families; FatA represents the commonly found 18:1 ACP TE, and
FatB includes TEs preferring acyl-ACPs having saturated acyl groups (2) . High amino acid sequence homology is found within each
family, and certain regions of the proteins are also highly conserved
between the two families ( (2) and see below). Despite the
similar enzymatic functions, the plant TEs share no protein sequence
homology with the animal and bacterial TEs(3) . Also, unlike
the animal and bacterial TEs, which have an active-site serine, the
plant TEs apparently utilize a catalytic cysteine since the enzymes are
sensitive to thiol inhibitors but not to serine hydroxyl-reactive
reagents(4, 5) . Davies et al.(5) also reported that the plant TE lost 97% of its
activity when treated with diethylpyrocarbonate, suggesting the
existence of an active-site histidine. These data are consistent with a
model in which a histidine as a general base enhances the thiol
nucleophilicity of a cysteine, thus enabling it to attack on the
thioester bond of the acyl-ACP substrate.
By constructing chimeric enzymes and site-directed mutagenesis, our earlier attempt in modifying the substrate specificity of a plant TE has gained some insights into the regions that are critical for substrate recognition(6) . However, due to the lack of crystal structure information, very little is known about the active-site residues that are essential for catalysis. To date, more than 30 plant TE sequences are available. This wealth of sequence information allows for the identification of amino acid residues that may be involved in catalysis or substrate binding by virtue of their conservation throughout this divergent set of enzymes. Sequence alignment of 33 plant TEs reveals one conserved cysteine and three conserved histidines. We mutagenized these residues to other amino acids in a 12:0 ACP TE (Uc FatB1) and demonstrated their possible roles in catalysis.
Figure 1: A partial alignment of the conserved histidines and cysteine. Only three representatives of each FatA and FatB type TE were selected for display. These residues are conserved throughout all 33 available plant TEs. Ct, Carthamus tinctorius (safflower); At, Arabidopsis thaliana; Bn, Brassica napus; Ch, Cuphea hookeriana; Cc, Cinnamomum camphorum (camphor); and Uc, Umbellularia californica (bay). The Uc FatB1 numbering is used.
The polymerase chain reaction conditions are as follows: five cycles were programmed with denaturation for 1 min at 94 °C, renaturation for 30 s at 48 °C, and elongation for 2 min at 72 °C. These first five cycles were followed by 30 cycles using the same program except with renaturation for 30 s at 60 °C.
Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis of the plant TEs(5, 8) . An amino acid sequence alignment of all available plant TEs (total of 33) reveals three conserved histidines and one conserved cysteine (Fig. 1). The Uc FatB1 is a good model for studying the critical roles of these amino acids because it is highly active and has been subjected to extensive biochemical study(3, 4, 5, 6) . In the absence of three-dimensional structural information, alanine-scanning mutagenesis can be used to identify structural determinants of catalytic reactivity of an enzyme. By site-directed mutagenesis, we have converted each of the three histidines to an Ala or an Arg and the cysteine to an Ala or a Ser. These mutants were expressed in E. coli, purified, and assayed for enzyme activity.
Mutation to
Ala at all four residues caused a substantial decrease of the enzyme
activity when compared with the wild-type Uc FatB1 (Table 1), indicating the functional importance of these
conserved amino acids. The specific activity of 9,010 µmol/min/mg
for the wild-type enzyme is about 80-900-fold higher than those
of most other TEs, e.g. specificity for a 14:0 ACP TE from Cuphea palustris is about 120 µmol/min/mg, and for the
8:0/10:0 ACP TE from the same species it is approximately 10
µmol/min/mg. ()Therefore, even with >90% loss of its
activity the bay TE is still as active as many other plant TEs, and the
activity can be easily detected in an in vitro assay. As shown
in Table 1, only C320A and H285A were completely inactivated,
whereas other Ala mutants displayed detectable activities. These
results demonstrate that Cys-320 and His-285 are essential for
catalysis.
We have also converted Cys-320 to a Ser. This mutant, C320S, retained considerable activity (approximately 60% of that of the wild type, see Table 1) but displayed different enzymatic characteristics. The activity of wild-type Uc FatB1 was sensitive to thiol reagents such as 5,5`-dithiobis(2-nitrobenzoic acid) and iodoacetamide (Table 2), a strong indication of the presence of a catalytic cysteine. Conversely, Uc FatB1 was insensitive to phenylmethylsulfonyl fluoride, a serine hydroxyl-reactive compound ( Table 2and (4) ). In contrast, the mutant C320S showed a reversed sensitivity, i.e. its activity was inhibited by phenylmethylsulfonyl fluoride but remained relatively insensitive to the thiol reagents (Table 2). In addition, consistent with the presence of an active-site cysteine, 1,4-dithiothreitol is essential for maximum activity of the wild-type Uc FatB1(4) . However, DTT is not required for the C320S mutant (data not shown).
Confirming a result previously reported(5) , treatment with
diethylpyrocarbonate resulted in about 90% loss of activity (Table 2), as would be expected for a cysteine enzyme typically
dependent on an active-site histidine. Our result of complete
inactivation by H285A demonstrates the likelihood of His-285 as a
proton acceptor for Cys-320 in catalysis. Supporting evidence for
His-285 being the active-site histidine also came from the optimal pH
data (Fig. 2). Here we show that the wild-type Uc FatB1
has a pH optimum of between 8.5 and 9.5, consistent with what was
reported previously(5) . On the other hand, mutant H285R shows
activity increases as pH increases from 7.0 to 12.3 and, very
interestingly, a significant burst of activity at pH >11. This
phenomenon can be explained as Arg-285 (pK 12) is
deprotonated at high pH, thus partially mimicking the role of His-285
in the wild-type enzyme(9) .
Figure 2:
pH
optima of the wild-type Uc FatB1 and mutant H285R. The
wild-type (open square) and mutant (solid square)
enzymes were assayed for 30 min at 30 °C in buffers with various pH
values containing 1 mM DTT, 0.01% Triton X-100, and 3
µMC-labeled 12:0 ACP using the procedures
described(4, 6) . Sodium acetate, Tris-HCl, and CAPS
buffers (100 mM) were used for the pH ranges 4.8-7.0,
7.0-9.0, and 9.0-12.3,
respectively.
Studies on TEs from bacteria,
animals, and plants have demonstrated that the enzymatic hydrolysis of
the thioester bond proceeds using the same basic catalytic machinery as
those of proteases and
lipases(5, 8, 10, 11, 12, 13) .
Crystal structure analysis of a myristoyl-ACP TE from the bacterium Vibrio harveyi has shown the catalytic triad of Ser, His, and
Asp(14) . While the active sites of the animal and bacterial
TEs resemble that of serine protease such as trypsin, for plant TEs
they are likely to be similar to that of papain, a plant cysteine
protease in which a thiol acyl enzyme is formed as a covalent
intermediate. A histidine residue acts as a general base catalyst
activating the cysteine as a nucleophile by partial proton abstraction.
Unlike the proteases, in which replacement of naturally occurring
active-site serine with a cysteine only produces poor enzymes with low K values toward natural substrates, the Ser
Cys mutants of animal TEs have been shown to be good catalysts,
retaining up to 90% of the activities(11, 12) . Here
we report that a Cys
Ser mutant of the plant TE is also highly
active (Table 1). We also show that His-285 is essential for the
nucleophilic attack by the cysteine, because the H285A is the only
histidine mutant that inactivates the enzyme. These observations
suggest that the Cys-320 of the wild-type enzyme and Ser-320 of the
mutant enzyme can attack, assisted by His-285, the thioester bond of
the substrate 12:0 ACP. At the present time no data are available for
the existence of an Asp or Asn residue forming the classic triad. There
are three Asp and four Asn, which are conserved in plant TEs. Although
a Ser-His-Asp triad has been found in the myristoyl-ACP TE of V.
harveyi, the main chain carbonyl of a Gln, instead of an Asp,
serves as a hydrogen bond acceptor for the active-site His in a similar
enzyme, the E. coli malonyl-CoA:ACP Transacylase(15) .
The mature proteins of both FatA and FatB classes of plant acyl-ACP
TEs are relatively conserved in length and sequence (approximately 80%
within each class and above 50% between classes) (2) . ()The cysteine and three histidines investigated in this
report are conserved in all plant TEs found to date. As shown in Fig. 1, the amino acids close to these four residues (especially
Cys-320 and His-285) are also highly conserved. In addition, no
insertion or deletion in the number of amino acids in the linear
polypeptide sequence is found between Cys-320 and His-285 in all
available plant TEs. This conserved linear relationship should be
reflected in a very similar three-dimensional structure. Therefore, we
believe that the results in this study have a general implication. It
is reasonable to hypothesize that all these TEs are structurally
similar because of their high sequence homology and similar functional
characteristics(16) . It is thus also reasonable to believe
that the active sites of all plant acyl-ACP TEs consist of the
homologues of Cys-320 and His-285. We can conclude that these plant
enzymes have active-site motifs of NQ(K)HN(S)N and YRR(K)ECG(Q/T), in
which the amino acids in parentheses occur relatively infrequently. In
fact, these two sequence motifs have been successfully used in
polymerase chain reaction amplifications of several TEs from various
plant sources (2) . The identification of the catalytic
cysteine and histidine in plant acyl-ACP TEs is a major step forward in
our continuous effort in engineering these important plant enzymes.