From the
The gene (ceo) encoding
N
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
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
It is
also apparent from Fig. S1that
N
To obtain greater expression of
N
Strains CEO571
and the N
The two
N
N
Our
observations with respect to the identification of amino acid residues
that participate in the binding of NADPH,
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
-(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.
-keto acid (e.g. pyruvate or
-ketoglutarate) and either the
- or
-NH
group of certain amino acids.
-(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) .
-(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.
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% NH
OH, 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
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 (-(CE)ornithine Synthase
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.
-(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.
-(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
N-(Carboxypropyl)amino
Acids
-(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(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
The sequence GSGNVAQGA which contains the GX
GXX
A motif of many (but not
all (52)) NADPH-binding -(CE)ornithine Synthase
-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
During
the original characterization of N-(Carboxypropyl) Derivatives
-(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/NH
OH/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 K
of 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.
-(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) .
-(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).
-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.
-(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
/EMBL Data Bank with accession number(s) U23376.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.