Báo cáo Y học: ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity potx

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,

Central Chemistry, Oxford, UK;
3
GlaxoSmithKline Pharmaceuticals, Worthing, West Sussex, UK
The clinically used beta-lactamase inhibitor clavulanic acid is
produced by fermentation of Streptomyces clavuligerus.The
orf6 gene of the clavulanic acid biosynthetic gene cluster in
S. clavuligerus encodes a protein that shows sequence
homology to ornithine acetyltransferase (OAT), the fifth
enzyme of the arginine biosynthetic pathway. Orf6 was
overexpressed in Escherichia coli (at  15% of total soluble
protein by SDS/PAGE analysis) indicating it was not toxic
to the host cells. The recombinant protein was purified
(to > 95% purity) by a one-step technique. Like other OATs
it was synthesized as a precursor protein which underwent
autocatalytic internal cleavage in E. coli to generate a and b
subunits. Cleavage was shown to occur between the alanine
and threonine residues in a KGXGMXXPX–(M/L)AT
(M/L)L motif conserved within all identified OAT
sequences. Gel filtration and native electrophoresis analyses
implied that the ORF6 protein was an a
2
b
2
heterotetramer
and direct evidence for this came from mass spectrometric
analyses. Although anomalous migration of the b subunit
was observed by standard SDS/PAGE analysis, which
indicated the presence of two bands (as previously observed
for other OATs), mass spectrometric analyses did not reveal
any evidence for post-translational modification of the b

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

catalysed by b-lactam synthetase (BLS/ORF3) [6–8].
Hydroxylation by clavaminic acid synthase (CAS/ORF5),
followed by hydrolysis of the guanidino side-chain catalysed
by proclavaminate amidino hydrolase (PAH/ORF4) yields
proclavaminic acid [9], which undergoes two further oxida-
tion steps, again catalysed by CAS, to produce bicyclic
clavaminic acid [10]. This is converted via an unknown
process involving epimerization at the C-3 and C-5 centres
to give (3R,5R)-clavaldehyde which is reduced, in a reaction
catalysed by an NADPH reductase (CAD/ORF9), to
clavulanic acid (reviewed in [1]).
In S. clavuligerus the genes for b-lactam biosynthesis are
grouped into a ÔsuperclusterÕ [11] in which the operons for
clavulanic acid and cephalosporin biosynthesis are adjacent.
The cephamycin gene cluster has been entirely sequenced
[12]. Including resistance, regulatory and transport genes, 11
genes have so far been reported in the clavulanic acid gene
cluster. Furthermore, the early clavam biosynthetic genes
are duplicated in a paralogous cluster responsible for the
biosynthesis of other clavams [13,14]. Thus, only functions
for the gene products of orfs2–5 in the early stages of the
pathway and the final stage (orf 9) of the clavulanic acid
gene cluster have been assigned (see above).
A
BLAST
search and sequence alignments indicate that
ORF6 displays 40% identity with ornithine acetyltransfer-
ase (OAT) from Thermus thermophilus and 30–36% identity
with OATs from other species [15,16]. OAT is one of the
enzymes involved in the biosynthesis of arginine from

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

and that catalysis proceeds via acetylation of the b subunit.
EXPERIMENTAL PROCEDURES
DNA manipulations were carried out by standard proto-
cols [22]. All chemicals were of reagent grade and obtained
from Sigma-Aldrich Co. Ltd. unless otherwise stated.
Restriction enzymes and IMPACT-CN System
TM
were
purchased from New England BioLabs Inc. Oligonucleo-
tides were synthesized by SigmaGenosys Ltd. Protein
concentrations were determined by the method of Bradford
[23].
PCR
The Orf6 gene was amplified by PCR from wild-type
S. clavuligerus genomic DNA (from GlaxoSmithKline) and
directly cloned into the PCR-Script
TM
vector (Stratagene).
The primers used were: forward primer, 5¢-ACGCT
CATATGTCCGACAGCACACCGAAGACG-3¢; reverse
primer, 5¢-CCATGTCCCTCCTGCCCTCGTCACCTTG
CAT-3¢. Orf6 was subsequently cloned into pET24a(+)
using BamHI/NdeI restriction sites. It was also cloned into
the pTYB12 and pTYB11 vectors of the IMPACT-CN
System
TM
using NdeI/EcoRI, and EcoRI/SapI sites, respec-
tively, to generate an intein-chitin binding domain fusion
vector.
Expression and purification of ORF6 in

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

containing 0.1% (v/v) Triton X-100. Following incubation
with column buffer containing 50 m
M
dithiothreitol for 16 h
at 23 °C to initiate cleavage of the intein tag protein, the
ORF6 was subsequently eluted with column buffer. ORF6
was judged to be > 95% pure by SDS/PAGE analysis
(17.5%). All protein samples were desalted, concentrated to
 4mgÆmL
)1
andstoredat)80 °C.
Gel filtration chromatography
The native molecular mass of purified ORF6 was
estimated by gel filtration using a Superdex 200 HR
column equilibrated with 100 m
M
Tris/HCl, pH 7.5. The
column was calibrated with ribonuclease A (13.7 kDa),
chymotrypsinogen A (25 kDa), ovalbumin (43 kDa) and
bovine serum albumin (67 kDa) (Gel Filtration Calibra-
tion Kit, Amersham Pharmacia Biotech.). An elution
volume parameter (K
av
) was calculated for each of the
calibration proteins and a calibration curve constructed.
By calculating K
av
for ORF6 the native molecular mass
was established.
Enzyme assays

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-

ethanol, 1 : 2]. The mixture was then vortexed for 15 s,
andheatedat95°C for 12 min. The absorbance at
470 nm was measured and the amount of ornithine
produced was determined by reference to a standard curve
of 0–1 lmol ornithine.
ORF6 kinetic studies were carried out in the presence of
1.0–6.0 m
M
N-acetylornithine and 6.0 m
M
glutamate. K
m
and the specific activity values were calculated using
SIGMAPLOT
(SPSS inc.).
The pH profile was determined using assay conditions as
above except with Tris/HCl buffer at pH 7.5–9.0, Mops
buffer at pH 7.0 and Mes buffer at pH 6.0–6.5, followed by
ninhydrin derivatization.
For
1
H NMR (500 MHz) assays, substrate concentra-
tions were doubled, and the amount of enzyme increased to
45 lg. The samples were incubated at 37 °Cfor2h,
lyophilized and resuspended in D
2
O. The
1
HNMR
(500 MHz) spectrum was recorded immediately.

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.

Gel electrophoresis
Both SDS/PAGE and native PAGE (17.5%) were per-
formed using standard techniques with a Bio-Rad Mini
Protean II kit. Samples were prepared by heating for 5 min
at 95 °Cwith2· SDS/PAGE sample loading buffer [0.635
M
Tris/HCl, 0.01% bromophenol blue (w/v), 10% SDS (w/
v), 50% glycerol (w/v), 1% 2-mercaptoethanol (v/v)].
Extended denaturation was achieved by heating with
2 · SDS/PAGE loading buffer at 95 °C for 30 min.
Mass spectrometric studies
Electrospray ionization mass spectra were recorded on a
Micromass BioQ II-ZS triple quadrupole mass spectro-
meter. Samples (10 lL) were introduced into the electro-
spray source via a loop injector as a solution with a final
protein concentration of 5 pmolÆlL
)1
in water/acetonitrile
(1 : 1, v/v) containing 0.2% formic acid.
For the nanoflow ESI mass spectrometry work, ORF6
samples were prepared at a concentration of 20 l
M
, in both
100 m
M
and 20 m
M
ammonium acetate, pH 7.0. For the
substrate selectivity experiments, samples of 20 l
M

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-

pressed, using both the pTYB12 and pTYB11 vectors, in
E. coli BL21 (DE3) cells as approximately 15% of the total
soluble protein by SDS/PAGE analysis. The pTYB vectors
allow for affinity purification of the target protein with
subsequent thiol-mediated cleavage of the tag in a single
step, utilizing a commercially avaliable modified intein
protein-splicing protocol. This single step purification using
the IMPACT-CN
TM
chitin bead column gave ORF6
protein samples of > 95% purity by SDS/PAGE analysis.
The pTYB12 vector introduced an additional three amino
acids (alanine, glycine and histidine) to the N-terminus of
ORF6 compared to that derived from the pTYB11 vector.
Subsequent activity analysis comparisons indicated that the
additional three amino acids had no affect on the functional
properties of ORF6. Almost identical CD traces were
obtained for the N-terminally extended and wild-type
proteins, indicating that the extra N-terminal residues did
not affect the gross structure of ORF6.
Autoproteolytic processing of ORF6
SDS/PAGE analysis of the purified ORF6 showed the
protein as two distinct bands, with approximate masses of
19 and 25 kDa (Fig. 1). From its gene sequence, ORF6 is
predicted to have a molecular mass of 42 kDa, and hence
ORF6 appeared to have undergone proteolysis, forming
two subunits. Only a trace band was sometimes present at
 42 kDa consistent with an efficient autocatalytic cleavage
in E. coli, which does not itself contain OAT [17]. The
N-terminal amino-acid sequence of the 19-kDa band (from

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-

ted that the b subunit of OATs from thermophiles run
anomalously by SDS/PAGE analysis [25] and the b subunit
of OAT from yeast has been observed as a double band [30].
To investigate this, denatured ORF6 was analysed by
electrospray ionization mass spectrometry, which indicated
that only a single b subunit species was present, with no
evidence for a post-translational modification as previously
suggested for the anomalous migration [25]. The mass
spectrum gave an a subunit mass of 18 816.2 Da and a b
subunit mass of 22 811.7 Da, which corresponds almost
exactly with the predicted masses of 18 815.4 and 22 810.3,
respectively, for a cleavage site between alanine 180 and
threonine 181. It seems likely that the ÔanomalousÕ obser-
vations for ORF6 result from the SDS/PAGE technique
and may reflect aggregation/incomplete denaturation. Con-
sistent with this, extension of the time the ORF6 protein
was heated with SDS before electrophoresis resulted in
apparent conversion of the lower b subunit band to the
upper band.
Native PAGE of the ORF6 protein suggested that it
exists as an ab heterodimer under these conditions.
However by gel filtration chromatography the molecular
mass was determined to be  84 kDa suggesting an a
2
b
2
stoichiometry. Presumably the interactions between the two
ab units are such that they are sufficiently weak to be
disrupted by electrophoresis conditions. It is possible that
yeast OAT, which has been reported to be an ab dimer, can

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
and (B) 20 m
M
ammonium acetate at pH 7.0 showing the
distribution of oligomeric species. Peak labels refer to the lower charge
states for the a and b monomers. Oligomeric species are indicated.
Experimental masses (n ¼ 6): a,18815.6±0.2Da;b,22810.4±
0.1 Da; heterodimer (ab), 41 629.1 ± 0.6 Da; heterotetramer (a
2
b
2
),
83 308.3 ± 11.0 Da.
Ó FEBS 2002 ORF6 from the clavulanic acid gene cluster (Eur. J. Biochem. 269) 2055
Kinetics and mechanism of ORF6
OATs catalyse the transfer of an acetyl group from
L
-N-acetylornithine to
L
-glutamate to form
L
-ornithine
and
L
-N-acetylglutamate. The presence of ornithine can be
detected by derivatization with acidic ninhydrin followed by
UV spectroscopic analysis, observing the absorption at
470 nm [24]. Using this assay it was possible to observe the
formation of ornithine from N-acetylornithine and gluta-
mate as catalysed by ORF6 (Fig. 3). The K

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.

forming ornithine) and reverse reactions in D
2
Oafter
quenching. Transfer of the acetyl group was shown to be
freely reversible. The equilibrium position (after a 2-h
incubation at 37 °C) for the (
L
-N-acetylornithine/
L
-gluta-
mate)/(ornithine/
L
-N-acetylglutamate mixture) was found
to be  3 : 2, respectively, by integration of the a-acetyl
CH
3
group of the N-acetylated compounds (Fig. 5).
The OAT from Chlamydomonas reinhardti is reported to
have
L
-N-acetylornithine hydrolase activity [32]. However,
incubation of ORF6 with
L
-N-acetylornithine in the absence
of a potential acetyl acceptor, led to no detectable obser-
vation (i.e. < 5%) of
L
-ornithine by the
1
HNMRand

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].

By analogy with other N-terminal nucleophile enzymes, the
nucleophilic site of the enzyme is the alcohol side-chain of
threonine 181, the N-terminal residue of the b subunit.
Direct evidence for the proposed mechanism came from ESI
mass spectrometry. For ORF6, using negative ion nanoflow
ESI mass spectrometry, three acetyl donors were examined
(
L
-N-acetylornithine,
L
-N-acetylarginine and
L
-N-acetylglu-
tamate). Negative ion spectra were recorded due to a lower
observed level of salt adducts compared to that present in
the corresponding positive ion spectra. Incubation of ORF6
with N-acetylglutamate followed by denaturation of the
protein indicated that approximately 37% of the b subunit
had undergone acetylation (Fig. 6). Some acetylation was
also observed for N-acetylornithine although at a lower level
(< 20%) than N-acetylglutamate (data not shown). Under
the same conditions, no acetylation of the b monomer was
evident when N-acetylarginine was employed as the sub-
strate.
Fig. 3. UV spectrum of ninhydrin derivatized reaction mixture at time
T ¼ 0andT ¼ 30 min.
Fig. 4. pH profile of ORF6.
Fig. 5.
1
H NMR spectrum of incubation of N-acetylornithine (NAO)

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

-a-amin-
oadipic acid,
L
-leucine, and
L
-alanine. With these substrates,
no ornithine was produced, indicating that the acetyl group
was not being transferred. However
L
-arginine,
L
-glutamine
and
L
-lysine did act as acetyl acceptors. After 20 min
incubation, the relative amounts of ornithine produced
using
L
-arginine and
L
-glutamine compared to
L
-glutamate
were 24% and 16%, respectively, as determined by the
ninhydrin assay. Lysine could not be measured as it
interfered with the ornithine derivatization conditions.
Activity was shown for all three by the
1
H NMR assay,
indicating the equilibrium position for the N-acetylorni-

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,

but not, for example, that of a-aminoadipate. It is
interesting to note that the side chain can contain basic,
acidic or neutral functional groups. Clearly X-ray crystal-
lography of ORF6 will be interesting, as there is currently
no 3D structural information available on any OAT.
Role of ORF6 in clavulanic acid biosynthesis
These results clearly demonstrate that ORF6 catalyses the
formation of
L
-N-acetylglutamate from
L
-N-acetylornithine
and
L
-glutamate, a key step in arginine biosynthesis, and are
the first experimental evidence for the functional assignment
of ORF6 from the clavulanic acid super-cluster. The first
step of the biosynthetic pathway to clavulanic acid
(Scheme 1, 1) involves the condensation of arginine with a
C-3 metabolite, glyceraldehyde 3-phosphate [4]. The role of
ORF6 may therefore be to increase the arginine available
for clavulanic acid production. Deletion mutant studies
show that when ORF6 production is blocked, clavulanic
acid production is completely halted in starch-asparagine
based medium, yet when grown in soy based medium
clavulanic acid production is approximately 40% of the
wild-type [13]. Jensen et al. have provided an explanation
for this observation by revealing that paralogous genes, not
directly responsible for clavulanic acid biosynthesis, are only
expressed in the soy based medium [13]. Another OAT in

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.

Ó FEBS 2002 ORF6 from the clavulanic acid gene cluster (Eur. J. Biochem. 269) 2057
There is no direct evidence for a role for multienzyme
complexes or channelling in clavam biosynthesis. However,
the lability (and likely toxicity) of some of the highly reactive
intermediates, especially 3R,5R-clavaldehyde, suggests that
intermediate channelling would be advantageous. Channel-
ing may also explain the selectivity problems posed by the
observation that ORF6 can accept arginine/N-acetylargi-
nine as substrates and the latter is a substrate for CAS. In
the case of other secondary metabolites, e.g. flavonoids [37]
there is evidence for channelling and protein–protein
interactions between ÔindividualÕ enzymes. ORF6 may
represent a link (possibly structural) between primary and
secondary arginine metabolism in S. clavuligerus. Finally,
because the full sequence of reactions in clavam biosynthesis
in S. clavuligerus has not been elucidated, another role for
ORF6 cannot be ruled out.
ACKNOWLEDGEMENTS
OCMS is jointly supported by the Biotechnology and Biological
Sciences Research Council, Engineering and Physical Sciences Research
Council and Medical Research Council. N. J. K. is supported by an
Engineering and Physical Sciences Research Council studentship and
HJM by a Biotechnology and Biological Sciences Research Council
CASE award. C. V. R. is supported by the Royal Society in a
University Research Fellowship. We thank R. T. Aplin for mass
spectrometric analyses and A. Willis for amino acid sequencing and
Prof. S. E. Jensen for encouragement.
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