©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Expression, Sequence Analysis, and Site-directed Mutagenesis of the Tn5306-encoded N-(Carboxyethyl)ornithine Synthase from Lactococcus lactis K1 (*)

Jacob A. Donkersloot (§) , John Thompson

From the (1) Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gene (ceo) encoding N-(carboxyethyl)ornithine synthase (EC 1.5.1.24) has been isolated from the sucrose-nisin transposon Tn5306 of Lactococcus lactis K1, sequenced, and expressed at high level in Escherichia coli. The cloned enzyme has allowed the synthesis of the novel N-carboxypropyl amino acids N-(1-carboxypropyl)-L-ornithine and N-(1-carboxypropyl)-L-lysine. Comparison of the deduced amino acid sequence of N-(1-carboxyethyl)-L-ornithine synthase (M = 35,323) to the functionally analogous octopine and nopaline synthases from crown gall tumors showed surprisingly little similarity. However, N-(1-carboxyethyl)-L-ornithine synthase and yeast saccharopine dehydrogenase exhibit homology at their N and C termini, which suggests that these two proteins constitute a distinct branch of the amino acid dehydrogenase superfamily. A centrally located 9-amino acid segment (GSGNVAQGA) in N-(1-carboxyethyl)-L-ornithine synthase is virtually identical with a sequence present in the -fold of the nucleotide binding domain of several microbial NADPH-dependent glutamate dehydrogenases. A much longer sequence of 80 residues has significant similarity to alanine dehydrogenase. Substitution of arginine 15 of N-(1-carboxyethyl)-L-ornithine synthase by lysine resulted in loss of enzyme activity.


INTRODUCTION

N-(Carboxyalkyl)amino acids (N-(CA)amino acids)¹(¹) have attracted attention by their unexpected relevance to many areas of biology, agriculture, and medicine (reviewed in Refs. 1 and 2). The enzymatic synthesis of these unusual compounds is catalyzed by members of the NAD(P)H-dependent reductase family (EC 1.5.1-) via the reductive condensation between the carbonyl moiety of an -keto acid (e.g. pyruvate or -ketoglutarate) and either the - or -NH group of certain amino acids.

Our introduction to this area of amino acid biochemistry stemmed from the discovery of two previously unknown N-(CA)amino acids in cells of Lactococcus lactis, an organism used widely as a starter in the dairy industry (3, 4, 5) . The synthesis of the compounds in question, N-(L-1-CE)-L-ornithine and N-(L-1-CE)-L-lysine, is catalyzed by a single enzyme, N-(CE)ornithine synthase (EC 1.5.1.24, Ref. 6), according to reactions i and ii (Fig. S1). To date, this enzyme has been detected only in certain strains of L. lactis(4, 7) , and little is known about the role of its products or of the structural and functional relationships between N-(CE)ornithine synthase and other N-(CA)amino acid synthases. Our interest in the evolutionary origin of N-(CE)ornithine synthase led, unexpectedly, to the isolation of a spontaneous mutant of L. lactis K1 that not only had lost N-(CE)ornithine synthase, but that was also unable to metabolize sucrose and to synthesize the lantibiotic nisin (8). Hybridization experiments indicated that the loss of these traits might have been due to the spontaneous excision of a transposon (Tn5306) from the chromosome (8, 9, 10) . In this paper, we first present physical evidence for this rare excision event, and, from comparative pulsed-field gel electrophoresis experiments, we also estimate the size of this large transposon.


Figure S1: Scheme 1



Inspection of Fig. S1 shows that reactions i and ii catalyzed by N-(CE)ornithine synthase are analogous to reactions iii-v that are mediated by saccharopine dehydrogenase (EC 1.5.1.7), D-octopine synthase (EC 1.5.1.11), and D-nopaline synthase (EC 1.5.1.19), respectively. Saccharopine dehydrogenase plays an important role in the metabolism of lysine in eucaryotic cells, and octopine synthase, nopaline synthase, and related oxidoreductases yield the ``opines'' found in crown gall tumors and in tissues of certain marine invertebrates (1, 2, 11, 12) .

It is also apparent from Fig. S1that N-(CE)ornithine synthase, saccharopine dehydrogenase, octopine synthase, and nopaline synthase all catalyze reductive condensations involving three substrates. Furthermore, the four enzymes are oligomeric and comprise subunits of comparable molecular mass (36-45 kDa). From these observations, we reasoned that cloning, site-directed mutagenesis, and comparison of N-(CE)ornithine synthase with the three previously sequenced (13, 14, 15, 16, 17) N-(CA)amino acid dehydrogenases (reactions iii-v), would provide insight to the catalytic, structural, and evolutionary relationships among members of this family of dehydrogenases.


EXPERIMENTAL PROCEDURES

Strains, Culture Conditions, and Transformations

The origin and maintenance of L. lactis subspecies lactis K1-23 and the N-(CE)ornithine synthase-negative mutant K1-42 have been described previously (7, 8, 9) . L. lactis ATCC 11454 and the sucrose- and nisin-negative derivative SLA2.8 were kindly provided by Dr. E. R. Vedamuthu (Microlife Technics). Strains C10, ML8, H1, and its plasmid-free derivative H1-4125 were gifts from Dr. G. P. Davey (New Zealand Dairy Research Institute). Escherichia coli strains Sure, TG1, and BMH 71-18 (mutS::Tn10) were obtained from Stratagene, Amersham, and Clontech, respectively. E. coli K38 containing pGP1-2 (18) was kindly provided by Dr. Stanley Tabor (Harvard Medical School). The E. coli strains and their derivatives were grown in L-broth with ampicillin, kanamycin, or tetracycline added whenever appropriate. Standard methods were used to propagate phage (19) . Transformations were done as recommended by Hanahan (20) .

DNA Preparation and Manipulations

Strains of L. lactis were grown at 32 °C in 100-500 ml of Lysis Broth (21) supplemented with 20 mMDL-threonine (22) until the absorbance (at 600 nm) reached 1.0. The cells were washed in TES buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, adjusted to pH 8.0) and incubated for 5 min at 37 °C in a solution containing 10% sucrose, 50 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, and 2 mg/ml lysozyme (23) . After lysis of the cells with SDS, genomic DNA was purified by a standard method (19). Recombinant plasmid DNA was isolated by the method of Birnboim and Doly (19, 24) . For sequencing, this DNA was further purified by polyethylene glycol precipitation (25) . Restriction site mapping, vector dephosphorylation, ligations, agarose gel electrophoresis, purification of DNA fragments from agarose, transfer of DNA to nitrocellulose, and subsequent hybridizations, were performed as described previously (8, 9, 19) .

Cloning of ceo

Genomic DNA (500 µg) from L. lactis K1-23 was incubated with 500 units of EcoRI for 2 h at 37 °C. The restriction digest was divided over three 10-40% (w/v) sucrose gradients (in 50 mM Tris-HCl, 5 mM EDTA, 1 M NaCl (pH 8.0)) and centrifuged for 18 h in a Beckman SW41 rotor at 35,000 rpm and 5 °C. Gradient fractions were tested for hybridization to the oligonucleotide Cos4 (see Hybridizations). Three µg of DNA that was highly enriched in the (20-kb) fragment encoding ceo was digested with 30 units of XhoI. After thermal inactivation of XhoI, 0.1 µg of this DNA was incubated (12 °C, 16 h) with 1 µg of the insertion vector Uni-Zap XR (Stratagene) and 1 unit of T4 DNA ligase in a final volume of 10 µl. The ligated DNA was precipitated with ethanol and dissolved in 10 µl of 0.2 TES buffer. Two µl of this preparation was incubated with a Gigapack II Gold extract (Stratagene), and the packaged phage was used to infect E. coli Sure. The plaques were screened for hybridization to Cos4 and for reactivity with antibody against N-(CE)ornithine synthase (see Immunoscreening). Several plaques that were positive in both assays were purified, and plasmid-containing derivatives were obtained by helper phage R408-mediated excision of the pBluescript SK insert (26) .

Hybridizations

Plaque lifts were prepared by placing a membrane filter (Millipore HATF) on top of the plaques in the agarose overlay. After 15 min of contact, the filters were air-dried (10 min) and placed successively for 3 min on filter paper pads soaked with denaturing solution (0.4 M NaOH, 0.6 M NaCl), neutralizing solution (0.5 M Tris-HCl buffer (pH 7.0), 3 M NaCl), and 2 SSC (1 SSC is 0.15 M NaCl, 15 mM sodium citrate (pH 7.0)). After baking (80 °C, 1 h), the filters were prehybridized (40 °C, 1 h) in 6 SSC, 5 Denhardt's solution (19), 0.5% SDS, 0.05 mg/ml (heat-denatured) sheared salmon sperm DNA. The hybridization was conducted at 40 °C for 16 h in a solution containing 6 SSC, 0.5% SDS, 1 Denhardt's solution, 0.1 mg/ml yeast RNA, and 0.6 µCi/ml P-labeled Cos4. After removal of the probe, the filters were washed five times at 40 °C with 6 SSC, 0.5% SDS and autoradiographed with intensifying screens for 10-20 h at 70 °C. The 39-mer Cos4 (5`-ATGAAAATTGGTCTTGTTAAAGCTAACTTTCCAGGTGAA) was synthesized by the phosphoramidite method, deprotected, and lyophilized. For the hybridizations, 20 pmol of Cos4 was end-labeled with [-P]ATP and T4 polynucleotide kinase (19) to a specific activity of 10 µCi/pmol. The 42-mer 5`-AAAGGAAATCCCTATGACAATGCAATGATGGAGTCTTTTTAT was used to detect IS904.This sequence was based on a (mostly) conserved segment (KGNPYDNAMMESFY) of the transposase encoded by this class of insertion elements (27) .

Immunoscreening

E. coli Sure was infected with recombinant phage, plated in an overlay, and incubated at 37 °C. After 6 h, when small plaques were evident, a membrane filter (Millipore HATF) was placed on top of the overlay and the plates were reincubated for an additional 14 h. The filters were rinsed for 1 h in PBST20 (0.15 M NaCl, 0.002 M phosphate buffer (pH 7.3), 0.05% (v/v) Tween 20 (28) ), and incubated for 2 h with antiserum (diluted 1000-fold in PBST20) against N-(CE)ornithine synthase (6) that had been absorbed (29) with E. coli Y1090 bound to Sepharose 4B (5 Prime - 3 Prime). The filters were washed four times in PBST20 and incubated for 2 h with alkaline phosphatase-conjugated anti-rabbit antibody (Promega; diluted 10,000-fold in PBST20) which had been absorbed with E. coli Y1090. After four washes, the filters were incubated with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

DNA Sequencing

The dideoxy chain termination method (30) with modified bacteriophage T7 DNA polymerase (31, 32) was used to sequence both strands of the XhoI-HpaI segment of p493 (Fig. 2, 3). Except for [-S]dATP (33) , the reagents were obtained as a kit (Sequenase version 2.0; United States Biochemical Corp.). Plasmid DNA was alkali-denatured (34) prior to annealing. The initial sequence data were obtained from a subclone (CEO502) containing a 0.7-kb DraI insert that hybridized to Cos4. Subsequent data were obtained by the primer-walking method. Inconsistencies or compressions were resolved by resequencing the relevant segments with 7-deaza-dGTP instead of dGTP (35) . Primers were synthesized by the cyanoethyl phosphoramidite method, eluted from the column with 30% NHOH, deprotected at 55 °C for 16 h, and passed through a NAP-10 gel filtration column (Pharmacia Biotech Inc.).


Figure 2: Restriction endonuclease map and ORFs of the p493 insert. The upper part shows various restriction endonuclease sites within the 7-kb EcoRI-XhoI insert, and the lower part shows an expanded view of the XhoI-HpaI segment that was sequenced in both directions. The closed bar represents the ceo coding sequence, and the open bars indicate other ORFs. The data obtained from the hybridizations with the Cos4 probe localized the N terminus of ceo (see text); the exact location of the C terminus was derived from the sequencing data.



Sequence Analysis

The DNA sequence was assembled, edited, and analyzed with the GCG (version 7) software package (36) . FASTA (37) queries of the Swiss-Prot data base at the EMBL were done via the Mail-FASTA program. BLAST (38) searches were performed via the NCBI BLAST E-mail server. In addition, the Smith-Waterman algorithm (39) and the BLITZ E-mail server were used to search the Swiss-Prot data base at the EMBL. The programs StemLoop and Terminator (40) were used to search for transcription terminators. The free energy of stem-loop structures was calculated according to the program Fold (41) with free energy parameters reported by Freier et al.(42) .

Expression and Analysis of N-(CE)ornithine Synthase

E. coli CEO493 was grown for 16 h in 500 ml of L-broth containing 50 µg/ml ampicillin. The cells were collected by centrifugation, resuspended in 30 ml of 0.05 M potassium phosphate buffer (pH 6.6) containing 1 mM dithiothreitol, and spun down again. The washed cells (3 g) were suspended in 3 ml of the same buffer and sonified (2 1 min) at 4 °C. After centrifugation (12,000 g, 30 min), the supernatant fluid was assayed for enzyme activity and protein content.

To obtain greater expression of N-(CE)ornithine synthase, plasmid p551 was constructed first by inserting the 1.5-kb HpaI fragment encoding ceo (Fig. 2) into pUC13. Cells containing p551 expressed 5-fold greater N-(CE)ornithine synthase activity when induced with isopropyl-1-thio--D-galactopyranoside. This finding established the orientation of the HpaI insert in p551. Plasmid p561 was obtained by inserting the p551 PstI-SstI fragment encoding ceo downstream from the T7 RNA polymerase promoter in pBluescript II SK. Transformation of p561 into E. coli TG1 carrying pGP1-2 (18) yielded strain CEO571.

Strains CEO571 and the N-(CE)ornithine synthase R15K mutant CEO641 (see below) were grown at 30 °C in 500 ml of medium containing ampicillin (50 µg/ml) and kanamycin (50 µg/ml), essentially as described by Tabor and Richardson (18) . When the absorbance (at 600 nm) of the culture reached 1.2-1.4, the temperature was raised to 42 °C to induce T7 RNA polymerase. After a 30-min induction period, the temperature was lowered to 37 °C, and rifampicin was added to a final concentration of 0.1 mg/ml. Cells obtained from 100-ml samples were washed in phosphate-buffered saline and stored at 20 °C. The frozen cells were suspended in 3 ml of 0.05 M potassium phosphate (pH 6.6) containing 1 mM dithiothreitol and sonified three times for 15 s with a microtip probe. The preparations were clarified by centrifugation (10,000 g), and the supernatant fluids were assayed. To obtain larger amounts of N-(CE)ornithine synthase, the procedure was scaled up to a 10-liter fermentor containing 7 liters of medium. The enzyme was purified to virtual homogeneity by DEAE-Sephacel and phosphocellulose-P11 chromatography and assayed as described previously (6). The protein content was determined by the Coomassie G-250 method (43). The SDS-PAGE procedure has been described (6, 7, 8

Enzymic Synthesis of N-(Carboxypropyl)amino Acids

N-(CE)ornithine synthase (2 units) was added to 1 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 5 mM -ketobutyrate (sodium salt), 5 mM NADPH, and either 2 mML-[U-C]ornithine or L-[U-C]lysine. After a 2-h incubation, N-(1-carboxypropyl)ornithine (0.27 µmol) and N-(1-carboxypropyl)lysine (0.15 µmol) were isolated and purified by previously described procedures (4, 5).

Pulsed-field Gel Electrophoresis

The method used was a modification of that described by Tanskanen et al.(44) . Washed cells of L. lactis strains K1-23 and K1-42 were encapsulated in 1% agarose plugs. Each plug was incubated successively at 37 °C with 1-ml amounts of: 0.2 TES buffer containing 1 mg/ml lysozyme (2 h), lysis buffer (6 mM Tris, 1 M NaCl, 0.1 M EDTA, pH 7.6, 0.5% Sarkosyl) containing 0.02 mg/ml RNase (2 h), lysis buffer containing 0.1 mg/ml proteinase K (16 h), 0.2 TES (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (2 h), and 0.2 TES (pH 8.0) for 6 h. The plugs were transferred to 0.2 TES (pH 8.0) and stored at 4 °C. For the restriction endonuclease digestions, a 3-mm slice was cut off and incubated for 16 h at 25-30 °C in 100 µl of buffer (20 mM Tris-HCl (pH 7.4), 5 mM MgCl, 50 mM KCl) containing 100 units of either SmaI or ApaI. These slices were inserted into the wells of a 1% agarose gel and subjected to pulsed-field gel electrophoresis in a CHEF DRII unit (Bio-Rad) for 16-20 h at 200 V with the switch interval ramped from 1-20 s. The electrophoresis buffer contained 45 mM Tris, 45 mM boric acid, 1 mM EDTA. In one run, the switch interval was ramped from 60-120 s to better separate the largest SmaI fragments.

Site-directed Mutagenesis

The change of Arg to Lys in N-(CE)ornithine synthase was carried out by the procedure of Deng and Nickoloff (45) . The oligonucleotide 5`-CAGGAAAGAAGATCTGAGCAAAAG (Clontech) was used to change the unique AflIII site in p561 to a BglII site, and 5`-CTGGAGAAAGAAAAGTCCCACTTC was synthesized to introduce the desired mutation. This latter primer was purified over an Oligonucleotide Purification Cartridge (Applied Biosystems). Both primers were phosphorylated enzymatically and annealed to heat-denatured p561. After second-strand synthesis and ligation, the mixture was used to transform the mutS strain BMH71-18. Plasmid DNA obtained from the pool of ampicillin-resistant transformants was incubated with AflIII and transformed into E. coli JM109. Restriction analysis, followed by sequencing, showed that about 40% of the transformants contained a plasmid with a BglII site as well as the desired mutation. Transformation of one of these plasmids (p561-1) into E. coli TG1 containing pGP1-2 yielded strain CEO641.

RESULTS

Mapping and Cloning of ceo

We have previously described a mutant (L. lactis K1-42) that lacks a number of genes, including ceo, due to the apparent excision of the nisin-sucrose transposon Tn5306 from the parental chromosome (8). To provide support for this hypothesis, the parent and the mutant genomes were probed for the presence of the transposase gene encoded by IS904 (the prototype of a group of insertion elements found in many strains of L. lactis(46, 47, 48) ). The results of this hybridization experiment showed that 1 of the 16 copies of IS904 in the parental chromosome had been deleted together with Tn5306 (Fig. 1A). To estimate the total size of this deletion and to map ceo, suitably restricted genomic preparations from the parent and the mutant were compared by pulsed-field gel electrophoresis. The ApaI restriction profiles consistently showed that a fragment of 235 kb was replaced by a 180-kb fragment in the mutant (Fig. 1B). The results obtained with SmaI confirmed that the size of the deletion (and of Tn5306) is 60 kb, because a 450-kb fragment of the parent was shortened to 380 kb in the mutant (Fig. 1C). To localize ceo on a fragment suitable for cloning, the 39-mer Cos4 was synthesized based on the N-terminal sequence of N-(CE)ornithine synthase (6) . This ``guessmer'' (49) hybridized strongly to two parental EcoRI fragments, but comparison with strain K1-42 indicated that ceo had to be located on the larger (20-kb) EcoRI fragment (Fig. 1D). Efforts to clone this fragment into the vector -GEM11 were unsuccessful, and we therefore attempted to clone the 7-kb EcoRI-XhoI fragment (Fig. 1D) in the phage vector Uni-Zap XR. About 5% of the plaques that were obtained reacted both with Cos4 and with antibody raised against N-(CE)ornithine synthase, and one of these plaques was used to obtain a plasmid-containing E. coli derivative. This isolate (CEO493) contained a plasmid (p493) with the expected 7-kb EcoRI-XhoI insert. A restriction map of p493 was constructed, and the approximate location of ceo on this map was derived from a series of Southern hybridization experiments with Cos4 as the probe (Fig. 2).


Figure 1: Location of ceo. A, hybridization of EcoRI-digested genomic DNA preparations to a gene probe for the IS904-encoded transposase. B, pulsed-field gel electrophoresis of ApaI-digested DNA. C, pulsed-field gel electrophoresis of SmaI-digested DNA. On the basis of relative intensities, one of the two 450-kb fragments in the parent is shortened to a 380-kb fragment in the mutant. The smaller panel shows that under optimal experimental conditions, the 450-kb doublet in lane 1 can be resolved. D, hybridization of the ceo probe Cos4. For all panels, lanes 1 and 3, L. lactis K1-23; lanes 2 and 4, L. lactis K1-42. The arrows point to the differences; all sizes are in kilobases.



Sequence of ceo

The 3.4-kb segment containing ceo was sequenced in both directions from the XhoI junction to the HpaI site downstream of ceo (Fig. 2). Examination revealed an open reading frame extending from nucleotide 2038 to 2976 (Fig. 3), that was preceded by a typical ribosome binding site (5`-AAGGA) centered nine nucleotides upstream from the initiator codon. The molecular weight of the predicted protein (M = 35,323) is in reasonable agreement with the M = 38,000 estimated for purified N-(CE)ornithine synthase by SDS-PAGE (6) . Significantly, the first 37 residues of the deduced protein are identical with the data obtained from the Edman degradation of the N terminus of the purified enzyme (6) . Two hexanucleotides which might constitute the ceo promoter are delineated in Fig. 3. The sequence of the putative ``35'' element (TTGTTA) of this promoter agrees with the respective consensus calculated from 21 lactococcal promoters (50) , but the putative ``10'' hexanucleotide (TATTAG) varies from the consensus by the presence of G (rather than A) at position 1964. This difference might account for the finding that N-(CE)ornithine synthase constitutes only 0.01% of the protein in cells of L. lactis K1 (6) . Analysis of the sequence downstream from the 3`-end of ceo revealed a potential transcription terminator with a stem of 16 and a loop of 7 nucleotides (G = 15.5 kcal/mol), about 250 nucleotides beyond the translational stop codon. The stem has six T residues at its 3`-end, which is typical of -independent transcription termination.


Figure 3: Nucleotide sequence of the L. lactis K1 XhoI-HpaI segment containing ceo. The deduced amino acid sequence of N-(CE)ornithine synthase is shown under the nucleotide sequence. Putative ``35'' and ``10'' promoter elements are boxed. The putative transcription initiation site is indicated with a dagger. The N-terminal amino acid sequence obtained by Edman degradation of the purified protein is underlined. The sequence motif of NADPH-binding -folds is doubly underlined. A possible transcription termination signal starts at nucleotide 3229. Putative starts of ORFs c2, b1, c3, and c4 (see Fig. 2) are indicated with bent arrows.



The 2-kb fragment upstream of ceo contains four open reading frames (c2, b1, c3, and d2) encoding hypothetical proteins of about 7, 20, 37, and 13 kDa ( Fig. 2and Fig. 3). No similarities that appeared to have biological significance were detected when these sequences were compared²(²) to entries in the NCBI and EMBL data bases.

Similarities among N-(CA)amino Acid Dehydrogenases

The programs Compare and DotPlot were used to detect similarities between N-(CE)ornithine synthase and each of the other three N-(carboxyalkyl)amino acid dehydrogenases that have been sequenced to date (13, 14, 15, 16, 17) . Graphic matrix analyses showed that N-(CE)ornithine synthase has surprisingly little overall similarity to either octopine synthase or nopaline synthase from Agrobacterium tumefaciens(³) Furthermore, the limited similarities between N-(CE)ornithine synthase and saccharopine dehydrogenase (from the yeast Yarrowia lipolytica) are restricted to the N- and C-terminal segments of these proteins³. Assessment of global similarities by use of the program Gap () showed that the sequence similarity between N-(CE)ornithine synthase and saccharopine dehydrogenase is unlikely due to chance (51) .

Dinucleotide Binding Domain of N-(CE)ornithine Synthase

The sequence GSGNVAQGA which contains the GX GXX A motif of many (but not all (52)) NADPH-binding -folds (53) was detected near the middle of N-(CE)ornithine synthase (Fig. 3; residues 171-176). Interestingly, eight of the nine residues of this sequence are identical (Fig. 4) with a segment in the putative NADPH binding domain of the glutamate dehydrogenases (EC 1.4.1.4) from E. coli(54, 55) , Salmonella typhimurium(56) , Neurospora crassa(57) , and Saccharomyces cerevisiae(58, 59) . These similarities, in conjunction with the recently reported x-ray crystallographic localization of the dinucleotide binding domain of glutamate dehydrogenase from Clostridium symbiosum(60) , permit identification of a segment within N-(CE)ornithine synthase that might interact with the ADP moiety of NADPH (Fig. 4). However, virtually no sequence similarity was detected elsewhere in these two, functionally similar, enzymes by matrix analysis³ (see also ).


Figure 4: Sequence comparison of the putative -folds within the nucleotide binding domains of N-(CE)ornithine synthase and two glutamate dehydrogenases. The alignments were calculated with the program BestFit. The secondary structure assignment of the C. symbiosum enzyme is based on its three-dimensional crystal structure (60).



Compared to glutamate dehydrogenase, one finds() more extended similarity between N-(CE)ornithine synthase and the alanine dehydrogenases from Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis (), and Mycobacterium tuberculosum(61, 62, 63) . The alignment from Asp-209 to Lys-290 of N-(CE)ornithine synthase revealed 15 identical residues and 35 additional conserved ones among these five enzymes (Fig. 5). The similarities of the segments from Lys-231 to Glu-251 and that from His-254 to Ile-291 of N-(CE)ornithine synthase and the related segments of the B. subtilis alanine dehydrogenase were unlikely due to chance (p = 1 10 and p = 8 10, respectively; these matches were also highly significant when analyzed by the Smith-Waterman algorithm.()


Figure 5: Alignment of N-(CE)ornithine synthase and alanine dehydrogenase. Identities are in reverse type, and similarities (based on a positive score in the PAM 250 matrix) are shaded gray.



Expression of ceo

The specific activity of N-(CE)ornithine synthase in an extract of strain CEO493 was 3-fold higher than that of a cell-free preparation of L. lactis K1 (). To allow the facile purification of larger amounts of enzyme for crystallization and functional studies (see below), strain CEO571 was constructed (18) . Enzyme assays showed that induced CEO571 cells had 460-fold higher N-(CE)ornithine synthase activity than L. lactis K1 (). After the temperature shift, N-(CE)ornithine synthase accumulated to become the major protein in these cells (Fig. 6A, lanes 1-4), but most of it was pelleted during centrifugation (at 10,000 g) of a sonicated cell preparation (Fig. 6A, lane 7). Sedimentation of the enzyme was not due to inefficient breakage of the cells or to mechanical trapping of the protein by cellular debris, because the subsequent sonication of the centrifugal pellet released little additional N-(CE)ornithine synthase (Fig. 6A, lane 8). Despite this loss of enzyme in inclusion bodies, the supernatant fluid obtained after centrifugation contained enough activity (Fig. 6A, lane 6) to allow the purification of considerable quantities of N-(CE)ornithine synthase. The simple 2-step procedure involving gradient DEAE-Sephacel and phosphocellulose P11 chromatography yielded 160 mg of virtually pure N-(CE)ornithine synthase from 59 g (wet weight) of cells (Fig. 6B).


Figure 6: SDS-PAGE analysis of expression, solubility, and purification of N-(CE)ornithine synthase. A, expression by E. coli CEO571 and solubility. Lanes 1-4, expression; lane 1, just before induction; lanes 2, 3, and 4, 60, 120, and 160 min, respectively, after temperature shift. Lanes 5-9, solubility of N-(CE)ornithine synthase; lane 5, after sonication; lane 6, supernatant fluid after centrifugation; lane 7, resuspended pellet; lane 8, supernatant fluid after a second sonication and centrifugation; lane 9, resuspended pellet after the second sonication and centrifugation. B, two-stage purification of N-(CE)ornithine synthase from E. coli CEO571. Lanes 1 and 2, preparation after DEAE-Sephacel chromatography (39 and 18 µg of protein, respectively); lanes 3 and 4, preparation obtained by phosphate gradient elution from phosphocellulose P-11 (25 and 8 µg of protein, respectively). The arrows point to N-(CE)ornithine synthase.



Enzymatic Synthesis of N-(Carboxypropyl) Derivatives

During the original characterization of N-(CE)ornithine synthase (6) , -ketobutyrate appeared not to be a substrate for the enzyme. However, in the presence of high levels of the cloned N-(CE)ornithine synthase (1-2 units/assay), a comparatively slow reductive condensation was detected between -ketobutyrate and either L-ornithine or L-lysine to yield the novel compounds N-(carboxypropyl)ornithine and N-(carboxypropyl)lysine, respectively. When compared to glyoxylate and pyruvate, the rate of formation of these N-(carboxypropyl) derivatives (0.3 µmol formed mg of protein min) from -ketobutyrate is only 1% of that of the corresponding N-(carboxymethyl) and N-(carboxyethyl) derivatives (26 µmol formed mg of protein min). The components of a mixture containing ornithine, lysine, and their N-(carboxyalkyl) derivatives could be resolved by two-dimensional TLC (Fig. 7).


Figure 7: Separation of N-(CA) derivatives of L-ornithine and L-lysine by two-dimensional TLC. Five µl of a mixture (containing 10 nmol of each compound) was applied to the cellulose 300 MN layer, and the components were resolved by sequential development in: Solvent 1, 1-butanol/acetone/NHOH/water (20:20:10:4 (v/v)), and Solvent 2, isopropyl alcohol/formic acid/water (20:1:5 (v/v)). The amino acids and their derivatives were detected with ninhydrin. Numbers 1, 3, and 5 refer to the N-carboxymethyl, -carboxyethyl, and -carboxypropyl derivatives of ornithine, respectively. Numbers 2, 4, and 6 indicate the corresponding N-(CA) derivatives of lysine.



Pyruvate Binding Site

A search of the data bases with the FASTA program revealed similarity between residues 11-40 of N-(CE)ornithine synthase and residues 87-119 of lactate dehydrogenase (EC 1.1.1.27) from B. subtilis (64). In the alignment shown in Fig. 8A, 14 amino acids are identical and 10 others represent conservative substitutions. Structure-function studies of lactate dehydrogenases have shown that Arg-91() greatly facilitates catalysis (65) , and the alignment suggested that Arg-15 of N-(CE)ornithine synthase might play an analogous role. To evaluate this possibility, this residue was changed to lysine by site-directed mutagenesis of p561. Transformation of the mutagenized plasmid into E. coli TG1/pGP1-2 yielded strain CEO641. SDS-PAGE revealed accumulation of full-length mutant protein after induction.³ However, an extract of induced CEO641 cells showed no enzymatic activity (), even when 14 times more of the R15K protein was added than in the CEO571 assay, or when the pyruvate concentration in the assay was increased to 12 mM (the Kof the native enzyme is 0.15 mM). The specific activity of the R15K mutant protein was calculated to be less than 0.5% of that of the native enzyme.


Figure 8: Alignment of the N termini of N-(CE)ornithine synthase, lactate dehydrogenase, and alanine dehydrogenase. A, N-(CE)ornithine synthase and lactate dehydrogenase from B. subtilis (64). The asterisk indicates the arginine residue involved in catalysis. B, N-(CE)ornithine synthase and alanine dehydrogenase from B. sphaericus (61).



DISCUSSION

The biosynthesis of N-(CE)ornithine and N-(CE)lysine is catalyzed by N-(CE)ornithine synthase, and previous results indicated that in L. lactis K1 the gene encoding this enzyme (ceo) is located on the sucrose-nisin transposon Tn5306(8, 9, 10) . In this paper we provide an estimate (60 kb) for the size of this element and demonstrate that this transposon may excise from the chromosome of the parental organism. The finding that an 60-kb fragment was lost due to the rare excision of Tn5306 (Fig. 1) is in reasonable agreement with the 70 kb of DNA inserted into the chromosome upon conjugal acquisition of other sucrose-nisin transposons (66, 67, 68) . The linkage between ceo and these transposons is certainly not universal, because sucrose-fermenting strains exist that lack N-(CE)ornithine synthase and vice versa (I). The possibility that ceo itself, perhaps together with adjacent genes of unknown functions (Fig. 2), may have inserted into Tn5306 should be considered.

The two N-(CE) derivatives of ornithine and lysine found in the amino acid pool of some strains of L. lactis(3, 4, 5) are, at present, the only such compounds reported for bacterial species. Our account of the cloning, expression, and sequence analysis of N-(CE)ornithine synthase provides the first description of this enzyme from any procaryotic organism. Glyoxylate, when used in lieu of pyruvate, yields the N- and N-(carboxymethyl) derivatives of ornithine and lysine, respectively (69) . By use of high levels of the cloned enzyme, and with -ketobutyrate as substrate, we now report the synthesis of the previously unknown N-(carboxypropyl) derivatives of ornithine and lysine. As illustrated in Fig. 7, these compounds are readily separated from the corresponding carboxyethyl and carboxymethyl analogs. These amino acid derivatives are potential inhibitors of enzymes such as -glutamyltransferase and -glutamylcyclotransferase (69) . The synthesis of the six N-(CA)amino acids by N-(CE)ornithine synthase augments the larger family of N-(CA) family of amino acids that includes the opines found in crown gall tumors of plants (2, 11) and in muscle tissue of certain species of marine invertebrates (1, 12) .

N-(CE)ornithine synthase from L. lactis appears to be unique because BLAST and FASTA searches of the 130,000 proteins presently catalogued in the data banks revealed remarkably few proteins with significant homology. Of the sequence similarities revealed by this analysis, that between N-(CE)ornithine synthase and alanine dehydrogenase currently appears to be the closest (; Fig. 5). Contrary to our initial expectation, the lactococcal enzyme exhibits remarkably little similarity to octopine synthase and nopaline synthase. This low degree of sequence similarity () may reflect the fact that the chirality of the products synthesized by these enzymes is distinct (4, 5, and see also Ref. 70).

Our observations with respect to the identification of amino acid residues that participate in the binding of NADPH, -keto acid, and ornithine (or lysine), can be summarized as follows.

NADPH Binding Domain

The sequence similarity between a segment of nine amino acid residues in both N-(CE)ornithine synthase and glutamate dehydrogenase from several microorganisms() was unexpected (Fig. 4). This conserved sequence contains the motif GX GXX A that frequently defines the dinucleotide binding domain of NADPH-dependent dehydrogenases (53, 71) . The two glycyl residues are close to the dinucleotide pyrophosphate moiety, and they define a sharp turn between the first -strand and the subsequent -helix of a conserved -fold (72) . From these considerations, and the recently published crystal structure of the NAD-dependent glutamate dehydrogenase from C. symbiosum(60) , a dinucleotide-binding -fold can be proposed for N-(CE)ornithine synthase (Fig. 4), but except for this segment, there is little similarity between the latter enzyme and glutamate dehydrogenase ().

Pyruvate Binding Site

Structure-function studies of L-lactate dehydrogenase (EC 1.1.1.27) have allowed the identification of specific residues required for pyruvate binding and catalysis (73) . Of these amino acids, Arg-91 enhances the rate of hydride transfer more than 1000-fold (65) , and the alignment (Fig. 8A) suggested that R15 of N-(CE)ornithine synthase might play an analogous role. Indeed, replacement of this residue with lysine yielded a catalytically inactive protein. Alanine dehydrogenase from B. sphaericus(61) , like lactate dehydrogenase, has relatively low overall similarity to N-(CE)ornithine synthase (23.4% identity). However, a region of stronger similarity is present at the N termini of these proteins (Fig. 8B), which suggests that this region may contain catalytically relevant residues. Notably, Arg-91 of lactate dehydrogenase is also conserved (as Arg-15) in alanine dehydrogenase.

Lysine/Ornithine Binding Site

No convincing similarities were detected when N-(CE)ornithine synthase was compared with other enzymes for which ornithine is a substrate. The closest relationship observed between members of the N-(CA)amino acid dehydrogenases was that between N-(CE)ornithine synthase and saccharopine dehydrogenase from the yeast Y. lipolytica (). Catalytically, there is also similarity between the two enzymes since, in the presence of high levels of lysine and pyruvate, saccharopine dehydrogenase (like N-(CE)ornithine synthase) can also form N-(CE)lysine (74) . The N-lysyl derivative synthesized by both enzymes exhibits L, L stereochemistry, and, therefore, some sequence and structural resemblances might be anticipated. The N termini of N-(CE)ornithine synthase and saccharopine dehydrogenase contain similar stretches of seven amino acids: DDQETSD and DDQEFVD, respectively. For N-(CE)ornithine synthase, this short sequence follows shortly after the putative pyruvate binding site, and one or more of the four acidic residues may interact with positively charged - or -amino groups of ornithine and lysine to facilitate catalysis.

Our analysis of the sequence data and the presumptive identification of functionally important residues suggest a number of site-directed mutagenesis experiments. Additionally, highly purified N-(CE)ornithine synthase is now available in quantities sufficient for x-ray crystallographic analysis and for structural comparison of this enzyme with other members of the amino acid dehydrogenase superfamily. Together, these genetic and physicochemical approaches may permit the unequivocal identification of active site residues and allow characterization of the functional domains of this trisubstrate enzyme.

  
Table: Global comparison of N-(CE)ornithine synthase to selected other dehydrogenases


  
Table: Expression of N-(CE)ornithine synthase activity


  
Table: Sucrose fermentation and presence of N-(CE)ornithine synthase in L. lactis



FOOTNOTES

*
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/EMBL Data Bank with accession number(s) U23376.

§
To whom correspondence and reprint requests should be addressed: National Institutes of Health, Bldg. 30, Rm. 316, 30 Convent Dr. MSC 4350, Bethesda, MD 20892-4350. Tel.: 301-496-4216; Fax: 301-402-0396; E-mail: donkersloot@nih.gov.

¹
The abbreviations used are: CA, carboxyalkyl; CE, carboxyethyl; kb, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis.

²
The programs BLAST and FASTA were used.

³
J. A. Donkersloot and J. Thompson, unpublished results.

The program BLAST (version 1.3) and the Blosum62 matrix were used.

The MPsrch program (version 1.3) was used via the BLITZ E-mail server at the EMBL.

The numbering relates to the B. subtilis enzyme; the corresponding residue in pig lactate dehydrogenase is Arg-109.

This segment is identical for the enzymes from E. coli, S. typhimurium, N. crassa, and S. cerevisiae.


ACKNOWLEDGEMENTS

We thank Robert J. Harr for dedicated technical assistance and Dr. E. Victoria Porter for help with computer problems. Drs. Stephen H. Leppla and R. Dwayne Lunsford offered constructive comments on the manuscript, and Drs. John Wootton and David Rice provided stimulating discussions related to glutamate dehydrogenase.


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