©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Characterization of a Heterologously Expressed Bifunctional Chorismate Synthase/Flavin Reductase from Neurospora crassa(*)

(Received for publication, June 2, 1995; and in revised form, June 26, 1995)

John M. Henstrand Nikolaus Amrhein Jürg Schmid (§)

From the Institute of Plant Sciences, ETH-Zürich, CH-8092 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Chorismate synthase (CS) (^1)(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.


EXPERIMENTAL PROCEDURES

Library Construction

Poly(A) RNA was prepared according to Sambrook et al.(13) from N. crassa mycelia that had been grown on Vogel's minimal medium N (14) at 25 °C. The cDNA was synthesized according to the instructions of the supplier (Stratagene). Double-stranded cDNA was ligated to EcoRI adaptors (Biolabs) and digested with XhoI. The cDNA was separated on a 0.8% agarose gel, the region corresponding to a size of 1-3 kilobases was excised, and the DNA was extracted. The size-selected cDNA was then ligated into the EcoRI-XhoI-cut pTrc99a-derived vector, pTrcEX. This vector was made by ligating the KpnI-EcoRI fragment, containing an XhoI site, of the multicloning site from Bluescript SK (Stratagene) into EcoRI-KpnI-digested pTrc99a (Pharmacia Biotech Inc.). Vector and cDNA ligation mixtures were electroporated into E. coli strain PLKF`(15) . Transformants were grown on Luria broth plates containing 100 mg/liter ampicillin, colonies were washed off, and plasmid DNA was extracted(15) . The resulting library consisted of 1.2 10^6 independent cDNA clones with average insert sizes of 1.2 kilobases.

Complementation of E. coli AB2849

The N. crassa cDNA library (1 µg) was used to electroporate cells of the CS-deficient E. coli strain AB2849(16) . One ml of Luria broth was added to 0.4 ml of electroporated AB2849 cells, and then the cells allowed to recover for 3 h at 30 °C with shaking. Fresh M9 medium was added to a final volume of 10 ml, and cells were centrifuged at 5000 g for 5 min. To the pellet 1 ml of M9 medium was added, of which 100-µl aliquots were spread each on 150-mm plates containing Dixon's medium (Life Technologies, Inc.) supplemented with 0.1 µM IPTG and 100 mg/liter ampicillin. Plates were incubated for 3-5 days at 25 °C until colonies were observed.

Expression Constructs

The full N. crassa CS coding sequence and derived constructs with deleted sequences representing amino acids Ile-Pro, Pro-Gln (Fig. 1), or both, are referred to as pTrc99a-NcCS, pTrc99a-NcCSDeltaC, pTrc99a-NcCSDeltaI, and pTrc99a-NcCSDeltaCI, respectively. NcCS, NcCSDeltaC, NcCSDeltaI, and NcCSDeltaCI refer to the corresponding protein products. All constructs were made with appropriate polymerase chain reaction and restriction fragments. Portions of constructs generated by polymerase chain reaction were verified by sequencing.


Figure 1: Nucleotide and deduced amino acid sequences of N. crassa CS cDNA.



Protein Purification

Cells of E. coli strain LC137 (htpR lon supC tsx::tn10), containing either pTrc99a-NcCS or pTrc99a-NcCSDeltaC, were grown in Luria broth at 25 °C to A = 1.0, and then IPTG was added to 1 mM. After 3 h, cells were harvested by centrifugation and stored at -20 °C until further use. Pellets were brought up in buffer A (50 mM Tris-HCl, pH 7.5, 0.4 mM dithiothreitol, 1.2 mM phenylmethanesulfonyl fluoride), and cells were lysed by sonication in the presence of 10 mg/liter lysozyme. The subsequent purification of CS activity was essentially performed as described by White et al.(8) except that dialysis against 2 liters of buffer A replaced negative DE-52 chromatography.

Gel Electrophoresis and Immunoblotting

Protein samples that had been precipitated in 6% trichloroacetic acid were brought up in 2 loading buffer and separated by SDS-PAGE (10%). Gels were either stained with Coomassie Brilliant Blue G-250 (15) or electroblotted onto nitrocellulose membranes with subsequent detection using rabbit antibodies raised against C. sempervirens CS essentially as described by Schaller et al.(17) .

Enzyme Assays

Continuous Assay

Unless otherwise indicated, activity of the bifunctional N. crassa CS was determined by continuous monitoring of the appearance of chorismate at 275 nm at 30 °C in a reaction volume of 0.5 ml containing 50 mM triethanolamine-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl(2), 200 µM NADPH, 10 µM FMN. Reactions were initiated by the addition of 80 µM EPSP.

End Point Assay

Due to the strong absorbance of dithionite at 275 nm, for comparison of CS activities in the presence of either dithionite or NADPH an end point assay had to be used. Reactions were performed in 0.25 ml containing 50 mM triethanolamine-HCl, pH 8.0, 50 mM KCl, 4 mM glutamine, 2.5 mM MgCl(2), 50 picokatals of anthranilate synthase, and either 200 µM NADPH or 5 mM sodium dithionite and initiated by addition of 80 µM EPSP. After incubation at 30 °C for 20 min the reaction mixtures were acidified with 25 µl of 1 N HCl and extracted with 1 ml of ethyl acetate, and anthranilate was measured spectrofluorometrically(12) .


RESULTS

Identification of N. crassa CS cDNAs by Complementation of CS-deficient E. coli Strain

The size-selected N. crassa cDNA library in the pTrcEX vector (see ``Experimental Procedures'') was used to transfect the CS-deficient E. coli strain AB2849. Two complementing plasmids were isolated and analyzed. Both cDNA inserts had common restriction digest fragments; therefore, they probably correspond to the same gene. The plasmid with the longest insert of 1.7 kilobases was designated pTrcEX-1.7. Both strands of cDNA insert were sequenced, and the sequence is shown in Fig. 1along with the deduced amino acid sequence. The complementing N. crassa cDNA was not in frame with the pTrcEX translation start. Since the predicted reading frame ends with a stop codon 5` relative to the first ATG of the CS coding sequence, it is assumed that reinitiation of translation occurred for expression of the reading frame encoding CS.

Similarity of Deduced Amino Acid Sequence to Known Monofunctional CSs

The deduced amino acid sequence of N. crassa cDNA was compared with other CSs and found most similar (79% similarity, 70% identity) to the S. cerevisiae CS (Fig. 2)(18) . The predicted molecular mass for N. crassa CS of 46.4 kDa is larger than those of three monofunctional CSs from B. subtilis, (^2)E. coli(19) and the predicted mature form of C. sempervirens(20) of 39.9, 38.1, and 41.7 kDa, respectively. This large difference between the sizes of N. crassa CS and other CSs can be accounted for by the presence of an internal region (amino acids Ile-Pro) along with a C-terminal domain (amino acids Pro-Gln), which have no counterparts in the other CSs. These regions were used as targets for subsequent deletion analysis.


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^2 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-NcCSDeltaC 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.



Expression of N. crassa CS in E. coli

For further characterization of the N. crassa CS encoded by the cloned cDNA, a new construct was made for expression in E. coli. In the expression construct, pTrc99a-NcCS, the N. crassa CS start codon was brought into optimal proximity to the pTrc99a promoter for expression of NcCS (see ``Experimental Procedures''). The heterologously expressed CS was essentially purified as described(8) . After Mono Q chromatography, fractions containing CS activity were pooled. The purification from 20 g of E. coli cells yielded 1.3 mg of protein with a specific activity of 140 nanokatals/mg protein. By SDS-PAGE a single prominent band was observed with a molecular mass of 50 kDa (Fig. 3A, Table 1). This band was also detectable with antibodies raised against C. sempervirens CS. The antibodies also cross-reacted with a band of similar mobility from a preparation of partially purified CS from N. crassa(9) (Fig. 3B). Kvalues for FMN and NADPH were determined to be 3.2 and 25 µM, respectively.


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).





The C-terminal Region and an Internal Region Are Not Essential for CS Activity

An internal region (amino acids Ile-Pro) and the C-terminal region (amino acids Pro-Gln) specific to N. crassa CS were assessed with respect to their importance for CS and/or FR activity. Constructs encoding N. crassa CS without the 18 internal amino acids, 29 C-terminal amino acids, or both, were made and designated pTrc99a-NcCSDeltaI, pTrc99a-NcCSDeltaC, and pTrc99a-NcCSDeltaCI. Theses constructs, including pTrc99a and pTrc99a-NcCS, were transformed into E. coli strain AB2849 and streaked out on either Luria broth or Davis' minimal media in the presence of 0.1 mM IPTG and 100 mg/liter ampicillin (Fig. 4). The full-length N. crassa CS cDNA as well as all truncated constructs conferred a CS phenotype after 3 days of growth at 25 °C, although cells lacking sequences encoding the 18 internal amino acids grew less vigorously. AB2849 cells harboring each construct were then grown in liquid culture under the same conditions used for NcCS expression (see ``Experimental Procedures''). In crude extracts, activity above background levels was only detectable from cells expressing either NcCS or NcCSDeltaC (data not shown).


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.''



NcCS with a Truncated C Terminus Is Still Bifunctional

In order to determine whether the C terminus of CS contained a NADPH-dependent FR domain, NcCSDeltaC was purified following essentially the procedure used for purification of the full-length enzyme. The purification protocol for NcCSDeltaC was not optimized; however, by very narrow selection of active fractions eluting from DE-52 and phosphocellulose chromatography columns, sufficient purified material was obtained (Table 2). A 3-ml fraction eluting from phosphocellulose yielded 87 µg of protein, consisting of NcCSDeltaC purified to apparent homogeneity and an apparent molecular mass of 45 kDa based on SDS-PAGE (Fig. 5). Using the continuous NADPH-dependent CS assay, the specific activity of purified NcCSDeltaC was determined to be 40 nanokatals/mg protein; this value is 3.5-fold lower than that of NcCS.



To determine whether the lower specific activity of NcCSDeltaC 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 NcCSDeltaC. Comparing NADPH- with dithionite-dependent CS activities in two independent experiments, however, NcCSDeltaC 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.




DISCUSSION

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(2) 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 NcCSDeltaCI and NcCSDeltaI 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-NcCSDeltaC 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, NcCSDeltaC 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 NcCSDeltaC 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 NcCSDeltaC 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. NcCSDeltaC 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.


FOOTNOTES

*
This work is supported by a grant from the Swiss National Science Foundation (to J. S. and N. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence should be addressed. Fax: 41-1-632-1084.

(^1)
The abbreviations used are: CS, chorismate synthase; EPSP, 5-enolpyruvylshikimate 3-phosphate; FR, flavin reductase; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis.

(^2)
D. J. Henner, sequence of Bacillus subtilis dbpA, mtr(A, B), ndk, che R, aro(B, E, F, H), trp(A-F), hisH, and tyrA genes. EMBL accession number M80245[GenBank].


ACKNOWLEDGEMENTS

We thank Dr. W. Rau (Botanical Institute of the University of Munich, Germany) for the N. crassa mycelia, Dr. J. Coggins (University of Glasgow, United Kingdom) for E. coli strain AB2849, and Dr. D. Rubli (ETH, Zurich) for excellent photographic assistance.


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