(Received for publication, June 2, 1995; and in revised form, June 26, 1995)
From the
Activities of all chorismate synthases (CS) so far analyzed are
absolutely dependent upon reduced flavin. For monofunctional CSs, which
represent the only class of CSs that have yet been cloned, the flavin
must be reduced either (photo-)chemically or by a separable flavin
reductase (FR) for in vitro activity. Neurospora crassa CS, in contrast, possesses an intrinsic FR activity and represents
the only firmly established member of a bifunctional class of CSs. To
better understand this bifunctional protein, a cDNA from an N.
crassa expression library encoding a 46.4-kDa protein was cloned
by complementation of the CS-deficient Escherichia coli strain
AB2849. The deduced amino acid sequence was highly similar (79%) to a
previously isolated Saccharomyces cerevisiae CS. The N.
crassa sequence was unequivocally shown to encode the bifunctional
CS/FR by analysis of the purified protein expressed in E.
coli. Based on sequence comparisons with known monofunctional CSs,
two regions of 18 internal residues and 29 C-terminal residues unique
to N. crassa CS were deleted, and the constructs were also
expressed in E. coli. The presence of these regions was found
not essential for complementation of the CS phenotype
of E. coli strain AB2849. Although a 3.5-fold decline in
specific activity of the purified CS from cells expressing the
C-terminal deletion construct was observed, bifunctional activity was
not eliminated. These data strongly suggest that the domain(s)
responsible for reduction of flavin lie(s) within regions in which
homology is also shared among monofunctional CSs.
Chorismate synthase (CS) ()(E.C. 4.6.1.4) catalyzes
the unusual 1,4-trans-elimination reaction from
5-enolpyruvylshikimate 3-phosphate (EPSP) to yield chorismate,
the last common intermediate in the synthesis of the three aromatic
amino acids phenylalanine, tyrosine, and tryptophan. The elimination
reaction likely proceeds by a stereochemically favored nonconcerted
route for which several models have been proposed. These include an
X-group mechanism(1) , an allylic rearrangement of phosphate
followed by a 1,2-elimination(2) , and a stepwise loss of
phosphate and a proton via a cationic intermediate(3) .
One interesting aspect of the reaction is the requirement for reduced flavin since there is no net redox change. Only a few quite diverse enzymes, the most familiar of which is acetolactate synthase(4) , require flavin and catalyze reactions not affecting redox state. To our knowledge, CS is the only one that requires reduced flavin. The precise role of reduced flavin in the reaction is unknown. However, recent evidence has indicated direct involvement of the flavin, based on spectrophotometric analysis of Escherichia coli CS where the formation of a possible flavin C(4a) adduct during single turnover experiments (5) and the appearance of a stable semiquinone radical in the presence of the inhibitor (6R)-fluoro-EPSP (6) have been shown. The rate-limiting C-(6-pro-R)-H bond cleavage and phosphate release appear to occur only after the formation of the flavin intermediate(7) .
CS is only active in the presence of reduced flavin, so it is not surprising that in some but not all species a flavin reductase (FR) activity co-purifies with CS. So far, no specific FR has been identified for CSs purified from either E. coli(8) or the higher plant Corydalis sempervirens(9) . In contrast, two polypeptides, NADPH-dependent FR and CS, respectively, co-purify as a single complex from Bacillus subtilis(10, 11) . However, the closest association between NADPH-dependent FR and CS activities is found in Neurospora crassa where both comprise a single bifunctional polypeptide(12) .
It is known that the bifunctional N. crassa CS has a larger molecular mass relative to monofunctional CSs. Therefore, the larger size of N. crassa CS was thought to be indicative of an additional NADPH-dependent FR domain. To test whether this is indeed the case and in order to better understand the nature of this bifunctional enzyme, a cDNA encoding N. crassa CS/FR was isolated. Sequences encoding either full-length or truncated N. crassa CSs were analyzed to define regions necessary for CS and/or FR activities.
Figure 1: Nucleotide and deduced amino acid sequences of N. crassa CS cDNA.
Figure 2:
Comparison of the deduced amino acid
sequence of N. crassa CS with the sequences of S.
cerevisiae CS and three known monofunctional CSs. Sequences of CS
from S. cerevisiae(18) , of the mature form of C.
sempervirens(20) , and of CSs of E. coli(19) and B. subtilis were aligned (see
``Experimental Procedures'') in descending order with respect
to similarity to the N. crassa protein. In boldfacetype are shown residues with identity to the N.
crassa sequence. The internal
(Ile
-Pro
) and C-terminal
(Pro
-Gln
) sequences as referred to in the
text targeted in deletion constructs (Fig. 5) are shown underlined and doubleunderlined,
respectively. Residues used for identification of a putative NADPH
binding fold and also shown in Fig. 6are labeled with asterisks. The sequence comparison was done with the program
PILEUP (GCG package, version 7).
Figure 5:
Purification of
pTrc99a-NcCSC products from E. coli. Protein
from sequential purification steps was separated by 10% SDS-PAGE. CE, crude extract (10 µg); AS, ammonium sulfate
(10 µg); DE, DE-52 chromatography (2 µg); PC,
phosphocellulose chromatography (1 µg); RP, Mono
Q-purified recombinant NcCS from E. coli (1
µg).
Figure 6:
Comparison of Pseudomonas putida todA (ferredoxin reductase in (24) ) and N.
crassa CS sequences. Sequences were aligned using BESTFIT (GCG
package, version 7). Boldfaceletters represent
residues that match ADP binding fold fingerprint
sequences(23) . Conserved glycine residues found in all
sequences used in generation of fingerprint are indicated by arrows. The N. crassa CS sequence shown is also
highlighted in Fig. 2.
Figure 3: Purification of N. crassa CS expressed in E. coli. Fractions from sequential purification steps were separated by 10% SDS-PAGE. CE, crude extract (10 µg); AS, ammonium sulfate (10 µg); DE, DE-52 chromatography, (2 µg); PC, phosphocellulose chromatography (2 µg); MQ, Mono Q chromatography (2 µg); NP, partially purified native CS from N. crassa mycelia (2 µg). Gels were either stained with Coomassie Blue G-250 (A) or immunoblotted with antibodies raised against C. sempervirens CS (B).
Figure 4: Complementation analysis of constructs containing internal and C-terminal deletions. Shown are constructs and results of complementation of E. coli strain AB2849 performed as described under ``Experimental Procedures.''
To determine whether the lower specific activity of NcCSC was due to a differential effect upon either the CS
or the FR activity, additional experiments using both NADPH- and
dithionite-dependent assays were performed (Table 3). With the
anthranilate synthase-coupled CS assay, the specific activities in the
presence of dithionite were higher than in the presence of NADPH for
both NcCS and NcCS
C. Comparing NADPH- with
dithionite-dependent CS activities in two independent experiments,
however, NcCS
C was more markedly affected (32 and 45%)
relative to NcCS (65 and 55%). As a control, the
monofunctional mature C. sempervirens CS (matCS in (21) ) was active only in the presence of dithionite.
Complementation of the CS-deficient E. coli strain AB2849 with a N. crassa cDNA library led to the identification of a cDNA encoding CS. The deduced amino acid sequence shows significant similarity to other CS sequences, including those of B. subtilis, C. sempervirens, and E. coli. These three CSs are monofunctional proteins, catalyzing chorismate formation in vitro only in the presence of reduced flavin. N. crassa CS, however, is the only CS to be so far clearly established as a bifunctional enzyme, possessing also an intrinsic FR, using NADPH to reduce FMN.
In order to better characterize the cloned bifunctional
CS, it was expressed in E. coli and purified to near
homogeneity. NADPH-dependent CS activity was not lost even after
extensive purification, confirming that the cloned cDNA encodes the
bifunctional CS. FR activity could not be measured directly either by
following the disappearance of NADPH or by appearance of reduced
2,6-dichlorophenol-indophenol, an artificial electron acceptor, in
purified preparations (data not shown), suggesting that the turnover of
FMN-H is low. However, since NADPH- dependent N. crassa CS requires FMN (K
= 3.2
µM) and since dithionite-reduced FMN is sufficient for CS
activity in the absence of NADPH, FR is by definition still present.
Molecular masses of the known monofunctional CS enzymes are smaller than that of N. crassa CS in a range from 4.7 to 8.3 kDa. Of all known CS sequences, that of N. crassa CS is most closely related to S. cerevisiae. Based on its size relative to other monofunctional CSs and its lack of any sequences similar to NADPH binding fingerprints, the S. cerevisiae CS was previously thought to be monofunctional(18) . On the basis of this homology between a bifunctional and a putative monofunctional CS, sequences were selected within N. crassa CS for deletion analysis to define domains responsible for FR activity. Our strategy was to remove two regions, one internal sequence of 18 amino acids, and 29 amino acids at the C-terminal end, to which no homologous sequence exists in the monofunctional CSs and which together account for the larger molecular mass of N. crassa CS relative to S. cerevisiae CS. These additional sequences were removed individually or in combination in an effort to selectively remove FR activity while maintaining CS activity. Based on complementation data all constructs still supported the growth of CS-deficient E. coli strain AB2849 on minimal medium, indicating that CS activity was at least partially intact with or without these two domains.
The
crude extracts of AB2849 cells containing the individual constructs
were tested for dithionite-dependent CS activity. No CS activity was
detectable from cells expressing constructs with the internal 18-amino
acid deletion. Although NcCSCI and NcCS
I
were sufficiently active for complementation, low CS activities in the
crude extracts made it impossible to draw any further conclusions with
respect to the role of the internal region of 18 amino acids on FR
activity. Since high CS activity in extracts of cells containing the
pTrc99a-NcCS
C construct was found, further purification
of the C-terminal deleted CS could be performed in order to determine
the deletion's effect upon bifunctionality.
After
purification, NcCSC retained bifunctional activity, which
then excluded the possibility that the FR activity exists as an
C-terminal fusion. This result along with the fact that the N-terminal
amino acid sequence of N. crassa CS aligns with monofunctional
CSs indicates that a simple translational fusion of distinct CS and FR
components is not likely to be responsible for bifunctionality. This is
obviously different from the linear arrangement of independent
activities exploited by other multifunctional proteins in the shikimate
pathway, a well characterized example being the pentafunctional arom complex from Aspergillus nidulans(22) .
Nevertheless, the specific activity of NcCS
C activity was
3.5-fold lower than that of NcCS using the NADPH-dependent
assay, in which case the deletion may have been inclusive of a region
affecting only FR activity. For this reason dithionite- and
NADPH-dependent activities were compared for both NcCS
C
and NcCS. The conclusion drawn from this comparison was that
there is no clear differential effect upon either FR or CS activity,
both being negatively impacted by the deletion (Table 3).
The
mature form of CS from the higher plant C. sempervirens and CS
from E. coli and B. subtilis, all monofunctional
enzymes, have molecular masses of 38-42 kDa. NcCSC
with a predicted molecular mass of 42.5 kDa, while still being
bifunctional, leaves little room for any independent FR domain
translationally fused N- or C-terminal to CS. This is further
emphasized by the fact that sequence homology is found between the
bifunctional CS and the monofunctional C. sempervirens CS up
to the region that was deleted. These observations point to a region
conferring NADPH-dependent FR activity embedded within a sequence
thought previously to be solely involved in CS activity.
Since there
is no FR domain fused N- or C-terminal to the CS encoding sequence, it
follows that NADPH binding domains must be located within sequences in
which homology is shared between monofunctional and bifunctional CS
enzymes. One region found, i.e. amino acids
Ala-Glu
did show some homology to an ADP
binding fold domain(23) , matching 7 out of 11 residues. Data
base searching using this domain yielded another putative NAD binding
domain from the ferredoxin reductase subunit of toluene dioxygenase,
the todA gene product (Fig. 6)(24) . No
similarly significant homology was found within amino acid sequences of
the monofunctional CSs. Whether this is indeed an NADPH binding fold
awaits further investigation.
Two primary conclusions can be drawn from N. crassa CS. The first is that the larger size of the N. crassa compared with monofunctional CSs is not a consequence of the presence of an additional FR domain. The second is that NADPH-dependent CS activity is probably not to be viewed as a later evolutionary event; rather, monofunctional CS activity can be also viewed as being a result of a loss of function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].