ORF6 from the clavulanic acid gene cluster of
Streptomyces
clavuligerus
has ornithine acetyltransferase activity
Nadia J. Kershaw
1
, Heather J. McNaughton
1
, Kirsty S. Hewitson
1
, Helena Herna
´
ndez
2
, John Griffin
3
,
Claire Hughes
3
, Philip Greaves
3
, Barry Barton
3
, Carol V. Robinson
2
and Christopher J. Schofield
1
1
Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, UK;
2
Oxford Centre for Molecular Sciences,

subunit. Extended denaturation with SDS before PAGE
resulted in observation of a single major b subunit band.
Purified ORF6 was able to catalyse the reversible transfer of
an acetyl group from N-acetylornithine to glutamate, but
not the formation of N-acetylglutamate from glutamate
and acetyl-coenzyme A, nor (detectably) the hydrolysis of
N-acetylornithine. Mass spectrometry also revealed the
reaction proceeds via acetylation of the b subunit.
Keywords: ornithine acetyltransferase; clavulanic acid;
N-terminal nucleophile hydrolase; arginine biosynthesis.
Streptomyces clavuligerus produces a number of b-lactams,
including clavulanic acid (Scheme 1, 1), which is a potent
inhibitor of serine b-lactamases and is clinically used in
combination with penicillin antibiotics [1,2]. Whilst a
synthesis of clavulanic acid has been achieved, the known
routes are low yielding and produce racemic material [2,3].
Thus, it is produced commercially by fermentation of
S. clavuligerus and as a result its biosynthesis has been of
considerable interest, particularly with respect to the
optimization of fermentation titres.
The biosynthetic pathway to clavulanic acid has been
partially elucidated (Scheme 1) [1,2]. It begins with the
condensation of arginine and glyceraldehyde 3-phosphate,
catalysed by ORF2, to produce 2-carboxyethyl-arginine [4].
It has been shown [5] that arginine is a later metabolic
intermediate than ornithine as, when the pathway from
ornithine to arginine is blocked, ornithine cannot be
incorporated into clavulanic acid. 2-Carboxyethyl-arginine
is cyclized to give the first formed b-lactam, deoxyguanidi-
noproclavaminic acid, via an ATP mediated ring closure

glutamate, in which ornithine is a key intermediate
(Scheme 2) [17]. Ornithine is produced from glutamate via
Correspondence to C. J. Schofield, Oxford Centre for Molecular
Sciences and The Dyson Perrins Laboratory, South Parks Road,
Oxford, OX1 3QY, UK. Fax: + 44 1865275654,
Tel.: +44 1865275625, E-mail: christopher.schofi
Abbreviations: OAT, ornithine acetyltransferase; CAS, clavaminic acid
synthase.
(Received 16 November 2001, revised 22 February 2002, accepted
25 February 2002)
Eur. J. Biochem. 269, 2052–2059 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02853.x
four N-acetylated intermediates, beginning with N-acetyl-
glutamate and ending with N-acetylornithine. In most
prokaryotes (with known exceptions of Enterobacteriaceae
and Sulfolobus solfataricus [17,18]) ornithine formation is
catalysed by OAT (also know as ARGJ), which transfers an
acetyl group from N-acetylornithine to glutamate. Hence,
once the pathway is initiated from glutamate and acetyl-
CoA by acetylglutamate synthase (ARGA), the acetyl
group can be recycled, thus feeding the first step of the
pathway. Some OATs are bifunctional, being capable of
forming N-acetylglutamate from acetyl-CoA and glutamate
[19–21]. Here we report studies on the orf6 gene product
(ORF6). They demonstrate that ORF6 has OAT activity
and provide mass spectrometric evidence that ORF6, and
by implication other OATs, exists as an a
2
b
2
heterotetramer

E. coli
The pTYB12/orf6 and pTYB11/orf6 plasmids were used to
transform E. coli BL21 (DE3) cells and grown at 30 °Cin
2TY media containing ampicillin at 100 lgÆmL
)1
. When the
D
600
reached 0.5–0.8, the temperature was lowered to 15 °C
and protein expression was induced by the addition of
0.3 m
M
isopropyl thio-b-
D
-galactoside. Following overnight
incubation, the cells were harvested by centrifugation at
14 333 g for 20 min at 4 °C. The cells were resuspended in
50 m
M
Tris/HCl, pH 7.5, and lysed by sonication. The
lysate was cleared by centrifugation for 10 min in a bench-
top centrifuge at 11 000 g and loaded onto a column of
chitin beads pre-equilibrated with column buffer (20 m
M
Tris/HCl pH 8.0, 0.1 m
M
EDTA, 2
M
NaCl). The column
was washed with 15 column volumes of column buffer

The ORF6 assay contained 100 m
M
Tris/HCl, pH 7.5,
6.0 m
M
N-acetylornithine, 6.0 m
M
glutamate (or other
putative substrate) and 30 lg of enzyme in a final volume
Scheme 1. Biosynthetic pathway leading to clavulanic acid. BLS, beta-
lactam synthetase (ORF3); PAH, proclavaminate amidino hydrolase
(ORF4); CAS, clavaminate synthase (ORF5); CAD, clavaldehyde
dehydrogenase (ORF9). The ÔunnaturalÕ CAS-catalysed hydroxylation
of
L
-N-acetylarginine to (2S,3R)-3-hydroxy-N-acetylarginine is shown
boxed.
Scheme 2. The role of OAT (ARGJ) in the biosynthesis of arginine.
ORF6 carries out the same reaction. The proposed acyl-enzyme in-
termediate is shown boxed.
Ó FEBS 2002 ORF6 from the clavulanic acid gene cluster (Eur. J. Biochem. 269) 2053
of 0.1 mL. Incubation was at 37 °C for 20 min. Ornithine
acetyltransferase activity was measured by modification of
the procedure of Chinard [24] which employs ninhydrin to
determine the presence of ornithine. Following incubation,
derivatization was accomplished by the addition of
0.4 mL MilliQ water and 0.5 mL ninhydrin reagent
[1.2
M
citric acid/1.5% (w/v) ninhydrin in 2-methoxy-

N-
L
-Acetylarginine for incubation with CAS was pro-
duced by the standard assay, containing 6.0 m
M
N-acetyl-
ornithine and 6.0 m
M
arginine, but the incubation time was
increased to 2 h. Following the incubation, ORF6 was heat
denatured at 95 °C for 5 min and precipitated protein
removed by centrifugation. To 50 lL of the supernatant
was added 40 lgofCAS,10m
M
FeSO
4
and 10 m
M
a-ketoglutarate, in a final volume of 0.1 mL. This was
incubated at 30 °C for 10 min. The products were detected
by HPLC, using a reverse-phase C
18
octadecylsilane column
(150 · 4.6 mm), monitoring absorbance at 218 nm. An
isocratic gradient of 10% methanol was used at a flow rate
of 1 mLÆmin
)1
. N-Acetylarginine and 3-hydroxy-N-acety-
larginine had retention volumes of 3.2 and 2.9 mL, respec-
tively.

ORF6
with a 20-fold molar excess of substrate in 20 m
M
ammo-
nium acetate, pH 8.0 were left at room temperature for
1–2 h. After this time, an aliquot was removed and partially
denatured by addition of an equal volume of acetonitrile.
Data were acquired using a quadrupole time-of-flight
mass spectrometer (QTOF I, Micromass UK Ltd, Altrin-
cham, UK). Positive ion spectra were recorded to compare
samples at different ionic strength and negative ion spectra
were used for the N-acetyl donors study. Caesium iodide
was used to calibrate the instrument over the mass range
100–10 000 m/z (manual pusher set at 180 ls) with an
acquisition step of 5 s. Samples were loaded into borosili-
cate capillaries, 1.0 mm outer diameter · 0.5 mm inner
diameter (Clark Electrical Instruments, Reading, UK),
which were drawn down to a fine taper and coated with
gold in-house. The capillary tip was cut manually under a
stereomicroscope to give the required diameter and flow. A
nitrogen backing gas line was used to initiate and maintain a
flow from the capillary. Nitrogen at room temperature was
also used as drying gas, and the ESI source was not heated.
RESULTS AND DISCUSSION
Expression and purification
Previously it has been reported that high-level expression of
OATs is problematic, with expression above a critical level
becoming deleterious for E. coli hosts [25,26]. Expression of
orf6 using the pET24a(+) vector resulted in production of
an insoluble 42-kDa protein. However, orf6 was overex-

pTYB11) was shown to be MSDSTPKTPR, identical to
that expected for the N-terminus of ORF6. This was
2054 N. J. Kershaw et al. (Eur. J. Biochem. 269) Ó FEBS 2002
consequently designated as the a subunit, the b subunit
being the larger C-terminal portion. The b subunit had the
N-terminal sequence TLLTFFATDA, which is consistent
with cleavage occuring between alanine 180 and threonine
181 of the KGVGMLEPDMATLL motif.
Processing of ORF6 into a and b subunits is consistent
with its assignment as an OAT. All 17 known (or putative)
OATs contain the motif KGXGMXXPX–(M/L)AT(M/
L)L, with a predicted post-translational cleavage site
between the alanine and threonine residues [27]. The alcohol
of the ÔunmaskedÕ threonine is believed to act as a
nucleophile which is acylated during the catalytic cycle of
OAT. Thus, OAT is a member of the family of N-terminal
nucleophilic enzymes [28,29]. Experimental evidence has
been reported for this cleavage site in OAT from three
thermophilic organisms Methanoccocus jannaschii, Thermo-
toga neapolitana and Bacillus stearothermophilus [25], which
were shown to undergo cleavage to form a and b subunits
following expression in E. coli.OATfromSaccharomyces
cerevisiae, has also been shown to cleave at this point and
evidence provided for an autocatalytic rather than a
protease-mediated process [30].
By standard SDS/PAGE analyses, the b subunit of
ORF6 was seen to consist of major (lower) and minor
(upper) bands with apparent molecular masses of
 25 kDa. Both b subunit bands gave identical N-terminal
sequences (TLLTFFATDA). Previously it has been repor-

also exist as a heterotetramer as for ORF6 and other OATs.
To provide direct evidence for the formation of a hetero-
tetramer we investigated the use of nanoflow ESI mass
spectrometry for samples under native solution conditions.
At a protein concentration of 20 l
M
in 100 m
M
ammonium
acetate solution at pH 7.0, the major species observed was
the a
2
b
2
heterotetramer (experimental mass: 83 308.3 Da,
calculated mass: 83 251.6 Da) consistent with the gel
filtration results. Charge states from the ab heterodimer
and monomers were prominent (Fig. 2A). Homodimers
were not detected at any significant level. At a lower
concentration of ammonium acetate (20 m
M
), the intensity
of the heterotetramer charge states decreased relative to the
monomers and the heterodimer was the major oligomer
observed (Fig. 2B).
Fig. 1. SDS/PAGE gel of protein purification. Lane 1, molecular mass
markers; lane 2, cell lysate; lane 3, purified ORF6. Note the presence of
two bands for the b subunit in the latter.
Fig. 2. Comparison of positive ion nanoflow ESI mass spectra of ORF6
in (A) 100 m

m
value for
L
-N-acetylornithine at 37 °C and pH 8.0 was estimated to
be 3.6 m
M
, with a specific activity of 87 nmolÆmin
)1
Æmg
)1
.
This K
m
value is similar to K
m
values for other OATs, which
include 0.5 m
M
for T. aquaticus [18] and 9.6 m
M
for
M. jannashi [25]. Maximal enzyme activity was between
pH 7.5–8.0, which is similar to pH profiles for other OATs
[25,31] (Fig. 4).
To confirm the identity of the ORF6 assay products and
to develop an assay that did not rely upon ornithine
detection, the crude reaction mixtures were characterized by
1
H NMR (500 MHz) spectroscopy after termination by
heat treatment. Spectra were obtained for both forward (i.e.

ninhydrin assays. Presumably, incubation of
L
-N-acetylor-
nithine results in reversible acetylation of the active site
threonine 181 [25] and deacetylation by water cannot
compete with that by
L
-N-acetylornithine.
Some OATs (e.g. that from Neisseria gonorrhoeae,
Bacillus stearothermophilus, S. cerevisae and B. subtilis) are
capable of forming N-acetylglutamate from glutamate and
acetyl-CoA [19,21,25,27]. Because the ninhydrin assay
monitors ornithine production it was not possible to use
this assay with acetyl-CoA and hence the reaction was
monitored by
1
H NMR. Within the sensitivity of this
assay (< 5% conversion on 1.0 mg scale) it was not
possible to detect any N-acetylglutamate production, and
therefore ORF6 appears to behave as a monofunctional
OAT. There are no obvious differences between the
amino-acid sequences that could account for the differ-
ence between monofunctional and bifunctional OATs.
However, the sequences of the monofunctional class
appear to have a slightly shorter N-terminus [33].
ORF6, which belongs to the monofunctional class, shares
this property.
Previously, with OAT from B. stearothermophilus and
T. neapolitana, radioactive studies have indicated that there
is a covalent acetylation of the enzyme during catalysis [25].

and various acetyl group acceptors with ORF6. The acetyl group of
NAO appears at 1.98 p.p.m. and is at lower field in all cases. The acetyl
groups for the acetylated acceptors appear between 1.98 and
1.96 p.p.m.
2056 N. J. Kershaw et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Substrate selectivity of ORF6
A series of alternative acetyl acceptors were also assayed
with ORF6, using
L
-N-acetylornithine as the acetyl donor.
D
-Glutamate was not a substrate and ORF6 selected
L
-glutamate as a substrate from racemic
D
,
L
-glutamate.
This indicates that ORF6 (and probably other OATs) could
be used as an alternative to the widely used amino-acid
acylases [34], in particular for the resolution of racemic
mixtures of certain amino acids with side chains containing
polar groups.
The side-chain selectivity of the acetyl-acceptor was
probed, assaying
L
-aspartate and
D
-aspartate,
L

thine:ornithine mixture was approximately 2 : 1 as deter-
mined by integration of the hydrogens of the a-acetyl CH
3
group of the N-acetylated compounds (Fig. 5). The obser-
vation that arginine acts as a substrate is particularly
interesting because
L
-N-acetylarginine is a good substrate
for CAS [10], being hydroxylated to give (2S,3R)-3-
hydroxy-N-acetylarginine (Scheme 1).
L
-N-acetylornithine
is a much poorer substrate for CAS [10]. A combination of
ORF6andCASmightbeusedtoprepare/ferment
3-hydroxy derivatives of arginine and ornithine that might
be useful as chiral starting materials for pharmaceutical
production. Incubation of ORF6 with N-acetylornithine
and arginine to produce N-acetylarginine, followed by
incubation with CAS and appropriate cofactors resulted in
the formation of 3-hydroxy-N-acetylarginine, as detected by
HPLC analysis. The observation that arginine/N-acetylarg-
inine can act as an acetyl acceptor/donor raises the question
as to how selectivity is achieved in vivo.
Kinetic studies have shown that OAT from B. stearo-
thermophilus operates via a ping-pong bi-bi mechanism in
which the enzyme only accepts the glutamate after the
ornithine has left the active site [25]. The selectivity and mass
spectrometric results are consistent with this and suggest
ORF6 has a side-chain binding site that can accommodate
the side-chains of ornithine, glutamate, lysine and arginine,

the arginine biosynthesis cluster of S. clavuligerus (ARGJ)
has also been identified [35], suggesting that the OAT role of
ORF6 is directed towards increased intracellular concen-
trations of arginine for clavam biosynthesis rather than
primary metabolism.
Because ORF6 is not the only OAT in S. clavuligerus it
may seem surprising that no clavulanic acid production was
observed for the orf6 deletion mutant in starch/asparagine
media [13]. Deletion mutants for other enzymes required for
arginine biosynthesis have been reported to affect clavulanic
acid production [36]. Thus, elimination of ARGC (an
enzyme involved in synthesis of ornithine from glutamate in
the arginine biosynthetic pathway) has been shown to
interfere specifically with the production of clavulanic acid
[36], albeit under different growth conditions.
Fig. 6. Acetylation of ORF6 b monomer monitored by negative ion
nanoflow ESI mass spectrometry. Spectra have been transformed on to
a mass scale. The average masses from three spectra are shown. The
experimental mass difference between the acetylated and nonacetylated
forms was 41.8 ± 0.1 Da (42.0 Da calculated difference). (A) 20 l
M
ORF6 in 20 m
M
ammonium acetate pH 8.0 (B) 20 l
M
ORF6 with a
20-fold molar excess of N-acetylglutamate in 20 m
M
ammonium
acetate pH 8.

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Ó FEBS 2002 ORF6 from the clavulanic acid gene cluster (Eur. J. Biochem. 269) 2059