Báo cáo Y học: A b-lysine adenylating enzyme and a b-lysine binding protein involved in poly b-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei pot

A b-lysine adenylating enzyme and a b-lysine binding protein involved
in poly b-lysine chain assembly in nourseothricin synthesis
in
Streptomyces noursei
Nicolas Grammel
1,
*, Kvitka Pankevych
2
, Julia Demydchuk
2
, Klaus Lambrecht
2
, Hans-Peter Saluz
2
,
Ullrich Keller
1
and Hans KruÈ gel
2
1
Max-Volmer-Institut fu
È
r Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie,
Technische Universita
È
t Berlin, Germany;
2
Department of Cell and Molecular Biology, Hans Kno
È
ll Institute
for Natural Product Research, Jena, Germany

Nourseothricins (syn. Streptothricins), a group of nucleoside
peptides produced by several streptomycete strains, contain
a poly b-lysine chain of variable length attached in amide
linkage to the amino sugar moiety gulosamine of the
nucleoside portion. We show that the nourseothricin-pro-
ducing Streptomyces noursei contains an enzyme (NpsA) of
an apparent M
r
56 000 that speci®cally activates b-lysine by
adenylation but does not bind to it as a thioester. Cloning
and s equencing o f np sA from S. noursei including its ¯ank-
ing DNA regions revealed that it is closely linked t o the
nourseothricin resistance gene nat1 and some other genes on
the chromosome possibly involved in nourseothricin bio-
synthesis. The deduced amino-acid sequence revealed that
NpsA is a stand-alone adenylation domain with similarity to
the adenylation domains of nonribosomal peptide synthe-
tases (NRPS). Further analysis revealed that S. noursei
contains a b-lysine binding enzyme (NpsB) of about M
r
64 100 which can be loaded by NpsA with b-lysine as a
thioester. Analysis of the deduced amino-acid sequence from
thegene(npsB) of NpsB showed that it consists of two
domains. The N-terminal domain of  100 amino-acid res-
idues has high similarity to PCP domains of NRPSs whereas
the 450-amino-acid C-terminal domain has a high similarity
to epimerization (E)-domains of NRPSs. Remarkably, in
this E-domain the conserved H-H-motif is changed to H-Q,
which suggests that e ither the domain i s nonfunctional or h as
a specialized function. The p resence o f one single adenylating

b-lysine activating enzyme in nourseothricin-producing
streptomycete and a separate binding protein suggests an
iteratively operating NRPS-module catalyses synthesis of
the poly b-lysine c hain.
Keywords: nonribosomal peptide synthetase; PCP-domain,
b-lysine, nourseothricin, Streptomyces.
The nours eothricins belong to the family of the s treptothri-
cin an tibiotics that are produced by various streptomycete
strains such as Streptomyces noursei [1]. These compounds
are nucleoside p eptides containing a carbamido-
D
-gulos-
amine core, to which a poly b-lysine chain and the unusual
amino acid streptolydine are attached in amide and
N-glycosidic linkages, respectively (Fig. 1). The various
members of the group differ in the length of their poly
b-lysine chains. Streptothricins are potent inhibitors of
prokaryotic protein biosynthesis, but are not used thera-
peutically due to their nephrotoxicity [2]. Nourseothricin is
currently being used under the name CloNat, and is an
effective selective agent for molecular cloning technologies
in fungi and plants [3±6]. On the other hand, stre ptothricins
are also being tested as fungistatics in agriculture for the
treatment of blast disease and other plant diseases [7].
Knowledge o f t he biosynthesis of streptothricin s m ainly
stems f rom in vivo precursor studies (Fig. 1; reviewed in
[8]). Thus, streptolydine is derived from arginine [9],
gulosamine from glucosamine [10], and b-lysine from
a-lysine [11]. As the nourseothricins combine the struc-
tural f eatures of p eptides and nucleosides, their bi osyn-

thesis involve s quite diverse enzyme a ctivities f or sugar
biosynthesis, p eptide bond formation, glycosylation and
the formation of the nourseothricin precursors such as
b-lysine and streptolydine. The poly (b-lysine) chains of
nourseothricins are unique structures as they are made up
from identical (b-lysine) amino-acid residues connected to
each other with e-(b-lysyl)-peptide bonds and with the
chain attached to the amino group of the gulosamine
moiety. Peptide bond formation in natural products is
often catalysed by nonribosomal peptide synthetases
(NRPSs), a family of highly conserved enzymes, which
are composed of modules each responsible for the
activation and incorporation of always one individual
Correspondence to H. Kru
È
gel, Department of Cell and Molecular
Biology, Hans Kno
È
ll Institute for Natural Product Research,
Beutenbergstraûe 11, D-07745 Jena, Germany,
Fax: + 49 3641 656694, Tel.: + 49 3641 656684,
E-mail: , or U. Keller, Max-Volmer-
Institut fu
È
r Biophysikalische Chemie und
Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Techni-
sche Universita
È
t Berlin, Franklinstrasse 29, D-10587 Ber lin, G ermany.
Tel.: + 49 30 314 25653, E-mail:

Abbreviations: NRPS, nonribosomal peptide synthetase; A-domain,
adenylation domain; PCP-domain, peptidyl carrier domain;
C-domain, condensation domain; 4¢-Ppan, 4¢-phosphopanthetheine;
E-domain, epimerization domain; M-domain, methylation domain.
*Present address: A ctin oDrug Pharmaceuticals GmbH, Hennigsdorf,
Germany.
(Received 7 June 2001, revised 11 October 2001, accepted 6 November
2001)
Eur. J. Biochem. 269, 347±357 (2002) Ó FEBS 2002
amino acid i nto a given peptide pr oduct [12,13]. The
sequential order and number of the various modules of a
NRPS system determines the sequence and the length of
the peptide product. The modules consist of domains
including the adenylation domain (A-domain) responsible
for amino-acid recognition and their activation as an
aminoacyl adenylate, and the peptidyl carrier domain
(PCP-domain or T-domain) C-terminal to the A-domain
providing a covalently bound 4¢-phosphopanthetheine
(4¢-Ppan) cofactor for thioester bin ding of amino-acid
substrates and of peptidyl intermediates [14]. The third
essential domain of a module is the condensation domain
(C-domain) located aminoterminally to the adenylation
domain which catalyses condensation of amino acid
thioester attached to adjacent modules. Besides the
minimal set of A, T and C domains, modules of NRPS
may also harbour epimerization (E) domains, methylation
(M) domains, cyclization (Cy) domains instead of the C
domain, oxygenation (Ox) domains or reduction (Red)
domains. These catalyse modi®cation reactions on amino
acids or peptidyl intermediates [15].

The fact that the poly b-lysine chains of the different
nourseothricins consist of identical residues raises the
question as to whether the residues are incorporated by a
modular peptide synthetase containing several distinct
b-lysine modules or whether there is only one module
condensing the various b-lysine residues iteratively. Another
question is how the b-lysines are added to the gulosamine
moiety. By analogy to some poly amino acids s uch as f olyl-
poly c-glutamate, an amino-acid polymer produced by
bacteria and eukaryotes [16], p oly b-lysine synthesis could
possibly occur by a mechanism involving activation as
b-lysyl phosphate and subsequent ligation of b-lysine
residues in an iterative fashion. Thus, the condensing
enzyme would belong to the ADP-forming amide bond
ligase superfamily containing enzymes such as folyl-poly
c-glutamate synthetase, UDP-N-acetyl-muramoyl-
L
-ala-
nine-glutamate ligase, glutathione synthetase or
D
-Ala-
D
-
Ala ligase. All of these condense carboxylate-containing
compounds with a free amino group without covalent
binding of the substrate to the enzyme. This differs from the
nonribosomal thiol template mechanism [17].
On the other hand, gene d isruption experiments in
streptothricin-producing S. rochei revealed a gene locus
involved in streptothricin biosynthesis with ®ve genes

including one encoding resistance against the antibiotic
[18]. One of the ORFs encodes a protein with similarity to
the adenylation domains of peptide synthetases. This points
to nonribosomal mechanisms of streptothricin biosynthesis.
However, from the data it was not clear which substrate the
enzyme would activate: b-lysine or streptolydine, the latter
containing an internal peptide bond.
To clarify the mechanism of poly b-lysine synthesis
during nourseothricin synthesis we set out to isolate the
hypothetical poly b-lysine synthetase or its NRPS equiva-
lent fro m S. noursei and to clone the gene. We found that
S. noursei contains a stand-alone A-domain that activates
b-lysine by adenylation. It was also found that S. noursei
harbours a protein that after activation speci®cally binds
b-lysine as a thioester . This protein contains a PCP-domain
and a second domain with strong similarity to E-domains of
peptide synthetases, which indicates that the poly b-lysine
chain of nourseothricins is s ynthesized by a thiol template
mechanism.
MATERIALS AND METHODS
Strains and their cultivation
S. noursei JA3890b was from the strain collection of the
Hans Knoell Institute [19]. The strain was maintained on
agar slants and cultivated in submerged cultures in m edium
M79 [20]. Mycelia for enzyme preparations were harvested
from cultures propagated in 500 mL c onical ¯asks contain-
ing 100 mL medium at 28 °C for 2±3 days. The cultures
were cooled on ice, the mycelia were collected by centri-
fugation at 4 °C, washed in 0.9% NaCl solution and stored
at ) 80 °C until use. S. lividans 1326, from the John Innes

Collection, was maintained and cultivated as described
previously [21].
Fig. 1. Structure o f nourseothricin a nd its biosynthetic precursors.
348 N. Grammel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Chemicals and radiochemicals
b-Lysine was kindly provided by U. Graefe, Hans Knoll
Institute
1
and by P. A. Frey, University of Wisconsin,
Madison, WI, USA. [
3
H]b-Lysine (220 Ciámmol
)1
)was
obtained from Hartmann Analytik, GMbH, Braunschweig,
Germany
2
.[
32
P]Tetrasodium pyrophosphate (17.8 Ciámol
)1
)
was from New England Nuclear (NEN). Streptolydine-
gulosamine was obatined by partial hydrolysis of nourseo-
thricin according to the previously described method [22].
The compound was characterized as described previously
[23]. All other chemicals were of the highest purity
commercially available.
Puri®cation of b-lysine activating enzyme
All operations were carried out at 4 °C in a cold room.

S. noursei mycelia (50 g) were suspended in 200 mL buffer B
and passed through a French pressure cell at 68 947 kPa.
The resultant homogenate was treated with  50 lgámL
)1
DNAse I (grade II, Sigma) in the presence of 20 m
M
MgCl
2
for 1 h. After centrifugation at 30 600 g
3
for 30 min, the
supernatant was applied onto a Q-Sepharose FF column
(column dimensions 10 ´ 3 cm) previously equilibrated
with buffer B (see below). After washing the column with
50 mL of buffer B, the enzyme was e luted with a 200-mL
linear gradient from 0 to 0.2
M
NaCl in buffer B (5 mL
fractions). Fractions with b-lysine-dependent ATP-pyro-
phosphate exchange activity were pooled and saturated
ammonium sulphate was added up to a ®nal saturation of
66%. T he solution was t hen l eft on ice fo r at least 2 h. Th e
resulting suspension was centrifuged as above, the pellet was
dissolved in buffer B and applied to a HiLoad 26/60
Superdex 75 pg column (Pharmacia), which had been
previously equilibrated with buffer B. The ¯ow rate was
1mLámin
)1
and the fraction size was 1 mL. Fractions
containing b-lysine activating activity were pooled.

The pooled enzyme was applied onto an anion exchange
column (Mono Q HR5/5, Pharmacia) equilibrated in
buffer B and was eluted with a linear gradient from 0 to
0.2
M
NaCl in buffer B (¯ow rate 1 mLámin
)1
,gradient
60 min). Fractions containing enzyme activity were po oled
and saturated ammonium sulphate solution was ad ded to a
®nal concentration of 10%. The mixture was applied onto a
phenyl Superose HR5/5 (Pharmacia) column equilibrated
with buffer B containing ammonium sulfate at 10%
saturation. The column was eluted with a descending
gradient of 10 to 0% ammonium sulphate (¯ow rate
0.5 mLámin
)1
, 45 mL total volume). The b-lysine-activating
enzyme eluted at an ammonium sulphate concentration
corresponding to 8±9% saturation. Fractions containing
enzyme were pooled and concentrated in a microconcen-
trator (Centricon 30, Amicon).
Concentrated enzyme was subjected to gel ®ltration
chromatography (Superose 12 HR 10/30 column, Pharma-
cia) using a Smart chromatography system (Pharmacia).
The ¯ow rate was 300 lLámin
)1
and 200 lL fractions were
collected (Fig. 2). Active fractions were subjected to SDS/
PAGE. After staining, the protein band corresponding to

the enzyme was isolated for further analysis.
Puri®cation of the b-lysine binding protein
All operations were carried out in a c old room. S. noursei
mycelia (40 g) suspended in 200 mL of buffer B was p assed
through a French pressure cell a t 68 947 kPa. DNAse I
(grade II, Sigma) was added at 50 lgáL
)1
and MgCl
2
at
20 m
M
and left on ice with stirring for 1 h. After centri-
fugation for 20 min at 30 600 g
4
, the supernatant was
adjusted to a conductivity of 4 mS w ith water (containing
10 m
M
dithioerythritol) and applied onto a Q-Sepharose FF
column (column dimension 10 cm ´ 3 cm) pre-equilibrated
with buffer B. After washing the column with 100 mL of
buffer B a linear gradient (total volume 200 mL) of 0±0.2
M
Fig. 2. Identi®cation o f NpsA by g el ®ltration
of b-lysine activating enzyme o n Superose 12.
Concentrate d enzyme (300 lL) from t he
phenlysuperose step of Table 1 were appl ied
onto a Superose 12 c olumn (Pharmacia).
300 lL fractions were collected. The frac-

tionation range from fraction 35±55 is shown.
(3/4) Absorbance at 2 80 nm; (bars) ac tivity
pattern of the b-lysine-de pendent ATP/PP
i
exchange. The inset shows S DS/PAGE (10%
polyacrylamide, according to [22]) of 30 lL
portions of the indicated fractions. St aining
was with Coomassie blue. The b and repre-
senting the b-lysine activating enzyme NpsA is
denoted b y an arrow.
Ó FEBS 2002 Poly b-lysine assembly in S. noursei (Eur. J. Biochem. 269) 349
NaCl was passed through the column and 5 -mL fractions
were collected.
Fractions were assayed by determination of covalent
binding of [
3
H]b-lysine to protein in the presence of the
b-lysine activating enzyme, ATP and MgCl
2
(see below).
Fractions containing b-lysine b inding activity were pooled
and saturated ammonium sulphate solution was added until
70% s aturation. After leaving on ice overnight, t he suspen -
sion was centrifuged as described above. Pelleted protein
was dissolved in a small volume of buffer B. The sample was
subjected to gel ®ltration using HiLoad 26/60 Superdex
75 pg column equilibrated with buffer B, and 1-mL
fractions were collected. Fractions containing the b-lysine
binding protein were combined and subjected to anion
exchange chromatography on Resource Q (6 mL c olumn,

Pharmacia) equilibrated with buffer B. A 120-mL gradient
(¯ow rate 2 mLámin
)1
) was applied and 2 mL fractions were
collected.
The active fractions were pooled and brought to 20%
ammonium sulfate saturation. The solution was applied
onto a phenyl Superose HR5/5 (Pharmacia) column
equilibrated with buffer B containing ammonium sulphate
at 20% saturation and the protein was eluted with a 60-mL
gradient (¯ow rate 1 mLámin
)1
) r anging from 20 to 0%
ammonium sulphate saturation. The enzyme was eluted at
6% saturation. Active fractions were pooled and concen-
trated to a ®nal volume of 200 lL using a Centricon 30
microconcentrator. The sample was then subjected to gel
®ltration on a Superose 12 HR 10/30 column (Pharmacia)
previously equilibrated in buffer C at a ¯ow rate of
200 lLámin
)1
. Fractions (300 lL) were collected and the
b-lysine binding protein containing fractions were pooled.
Active fractions were subjected to analysis by gel electro-
phoresis.
Enzyme assays
The ATP-pyrophosphate exchange reaction mixture con -
tained 1 m
M
b- lysine, 2.5 m

M
ATP, 5 m
M
MgCl
2
,0.1m
M
tetrasodium pyrophosphate and 2 ´ 10
5
c.p.m. [
32
P]tetra-
sodium pyrophosphate and 10±50 lLofb-lysine a ctivating
enzyme fraction in a total volume of 220 lL. The mixture
was incubated for 10 min at 28 °C and stopped by the
addition of 0.5 mL charcoal suspension [24]. After 10 min
on ice, the charcoal was collected by suction ®ltration on
glass ®ber ®lters, washed once with 35 mL of water and
after drying at 80 °C (1 h), the ®lters were counted in a
liquid scintillation counter. Speci®c activity is de®ned as
nkatal, the amount of enzyme catalysing the incorporation
of 1 nmol pyrophosphate into ATP per second in the
presence of b-lysine.
The b-lysine binding protein was assayed in a coupled
assay with the b-lysine activating enzyme. The assay
contained 1±2 pkatal of b-lysine activating enzyme, 0.1 m
M
b-ly sine, 0.1 lCi [
3
H]b-lysine, 18 m

M
ATP, 33 m
M
MgCl
2
and 5±25 lLofb-lysine binding protein fraction in a total
volume of 60 lL. After 30 min of incubation at 28 °C,
2 m L 7% trichloroacetic acid was added. The mixture was
left on ice for 30 min The precipitated protein was
collected on membrane ®lters (ME 30, Schleicher &
Schuell), washed with 35 mL of water and after drying,
radioactivity was counted in a liquid scintillation counter
[25].
Buffers and solvent systems
Buffer B contained 0.1
M
Tris/HCl,pH8.0,4m
M
dithio-
erythritol, 1 m
M
benzamidine, 1 m
M
phenylmethylsulfonyl
¯uoride, 2 m
M
EDTA. Buffer C was the same as buffer B
except that it contained 0.05
M
Tris/HCl, pH 7.5. Solvent

systems for thin layer chromatography of b-lysine were
n-butanol/aceticacid/water (4 : 1 : 1,v/v/v;solvent system I)
or isopropanol/acetic acid/water (7 : 3 : 2 , v/v/v; solvent
system II ).
Methods of analysis
Protein concentrations were determined according to
Bradford [26]. SDS/PAGE was carried out according to
Laemmli [27]. Staining of gels was according to standard
procedures. Radioactivity determinations were by scintilla-
tion counting with a scintillation cocktail (Quicksafe A,
Zinsser Analytic) [25]. Thin-layer chromatograms (Silica gel
60, Merck, Darmstadt) were autoradiographed by exposure
to Kodak X-ray ®lm ( Biomax MS). b-Lysyl thio ester was
analysed by performic acid treatment of trichloroacetic acid
precipitated enzyme thioester as described previously [25].
Protein sequence determinations
Peptide sequences were determined with a Procise peptide
Sequencer (Applied Biosystems). Bands from SDS/PAGE
separations of the b-lysine activating enzyme blotted onto
poly(vinylidene di¯uoride) membrane were visualized with
Ponceau S , cut out and directly sequenced. In the case of the
b-lysine binding protein, bands in gels were visualized by
Coomassie staining. Gel pieces were cut out and subjected
to in-gel digestion with t rypsin as described previously [28].
After elution, the tryptic peptide mixture was separated by
HPLC (lRPC C2/C18 column, Pharmacia) with acetonit-
rile/water gradients in the presence of tri¯uoroacetic acid.
Well resolved peaks were subjected to sequencing. In the
case of the b-lysine binding protein six peptide sequences
were obtained which were used for the design of various

oligonucleotide primers for PCR The pair pcp 15, GAG
CACGGCMGRGAGGAGGC/PCP; 6, SGCSARGTG
SCCSACSGT gave a clone encoding a partial sequence
of NpsB.
DNA manipulations
All DNA manipulations were performed according to
published procedures [29]. In particular, genomic DNA of
S. noursei was prepared from lysozyme-digested mycelium
by phenol/chloroform puri®cation as described previously
[21]. To construct a genomic library, the DNA was partially
digested with Sau3A. DNA fragments in the size range from
10 to 20 kb were ligated to BamHI-cleaved lambda phage
vector arms (Lambda GEM-11 Packagene system, Pro-
mega). Screening for recombinant phages carrying nour-
seothricin biosynthesis genes was initially carried out by
hybridization of p laques with the nourseothricin resistance
gene nat1 [4±6], a nd in the later course of this work, a
fragment of the b-lysine-binding enzyme gene was used. The
latter fragment had been generated by PCR from chromo-
somal DNA of S. noursei using primers derived from two
internal peptide sequences of the b-lysine binding protein.
350 N. Grammel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The labelling of the probes was with the digoxiginen-
labelling kit from Boehringer, Mannheim. Hybridizing
plaques were picked, puri®ed and analysed by restriction
mapping and hybridization along with chromosomal DNA
as control. From each screening, one representative phage
(phage ph1-2 and phage ph41, respectively) was chosen for
subclone preparation using pUC118 or pBlueScriptKS.
Sequencing was performed on a LiCor automated system.

Plasmids
The plasmid for expression of npsA was pDW5 (K. Weber,
J. Demydchuk, U. Peschke, unpublished results). Plasmids
for subcloning and sequencing were pUC118 and pBlue-
ScriptKS.
Nucleotide sequence accession number
The DNA sequence data have been deposited in the EMBL
nucleotide sequence database under accession nos.
AJ315729 (npsA) and AJ315730 (npsB).
RESULTS
b-Lysine activation in
S. noursei
Peptide synthetases activate their amino-acid substrates by
adenylation w ith subsequent covalent binding to a PCP via
a thioester linkage. To seek b-lysine adenylating activity
possibly involved in nourseothricin biosynthesis, protein
extracts of S. noursei actively synthesizing nourseothricin
were fractionated by anion exchange chromatography on
Q-Sepharose FF matrix (Pharmacia). Assay of fractions
from such separations for b-lysine-dependent ATP-pyro-
phosphate exchange revealed a single peak of enzyme
activity (not shown). The enzyme was further puri®ed by gel
®ltration on Superdex 75 pg, anion exchange chromato-
graphy on Mono Q HR5/5 and hydrophobic chromatog-
raphy on phenyl Superose. In all these separations the
activity was found in one single peak. The ®nal puri®cation
was 50-fold (Table 1).
SDS/PAGE analysis of enzyme from the last puri®cation
step revealed several other protein bands (not shown). As
further attempts t o purify the enzyme activity to homoge-

neity failed, we subjected the enzyme from the phenyl
Superose step to gel ®ltration on Superose 12 HR. The
activity of each fraction was determined and each fraction
was subjected to SDS/PAGE analysis. By this procedure,
activity could be c orrelated with the intensity of a particular
band of 56 kDa (Fig. 2). The enzyme was named NpsA
(nourseothricin peptide s ynthetase A). Microsequencing of
this band yielded the N-terminal sequence: MESS
ASSFLEPFFDVXR.
Characterization of the b-lysine activating
enzyme (NpsA) from
S. noursei
Passing NpsA through a calibrated Superdex 75 pg gel
®ltration column revealed that the enzyme has an M
r
between 58 000 and 60 000. This ®tted with the estimated
molecular mass o f NpsA in its denatured form (Fig. 2) and
also indicates that the native form of the enzyme is a
monomer. The puri®ed enzyme when incubated with
tritium-labelled b-lysine, ATP and MgCl
2
did not bind the
labelled amino acid covalently, which indicates that the
enzyme most probably represented a stand-alone A-domain
without a PCP-domain.
The enzyme's substrate speci®city was determined by
measuring the ATP/pyrophosphate exchange in the pres-
ence of different amino acids structurally related to b-lysine.
The enzyme did not activate a-lysine or arginine (b-a rginine
not tested). This indicates that the active site of the e nzyme

can strictly distinguish between an a-amino and a b-amino
group of lysine. O ther b-amino acids such as b-alanine or
b-aminobutyric acid, c-aminobutyric acid and e-amino
caproic acid were not activated. Thus, the activating enzyme
appears to be strictly speci®c for b-lysine . The strict
speci®city of the enzyme for b-lysine strongly suggests that
it is p art of the nourseothricin synthesizing enzyme system.
No other b-lysine activating enzymes were detected in
S. noursei.
Cloning of the
NpsA
gene
Phages p h41 and phN6 are overlapping clones obtained
from screening o f a phage library of S. noursei DNA using
the nourseothricin resistance gene nat1 as a probe (Fig. 3).
nat1 has been previously cloned from S. noursei by its
property to confer resistance to nourseothricin in foreign
streptomycetes [4]. The gene encodes a nourseothricin-
acetylase (NatI) which speci®cally monoacetylates the
b-lysine chain of nourseothricin which makes this com-
pound antibiotically inactive. It is known that most, if not
all, antibiotic biosynthesis gene clusters contain resistance
genes against their own antibiotic [31]. We therefore
concluded that the gene for the b-lysine a ctivating enzyme
Table 1. Puri®cation of the b-lysine activating enzyme NpsA f rom Streptomyces noursei. Cells (50g) from a 72-h culture of S. noursei was used.
Puri®cation w as based on A TP-pyropho sphate e xchan ge dependent on t he presence of b-lysine. On e nkatalámol
)1
is the a mount of enzyme
catalysing the exchange of 1 n mol of pyrophosphate into ATP per s econ d. ND, not determined.
Puri®cation

step
Volume
(mL)
Protein
(mg)
Activity
(nkatal)
Speci®c activity
(pkatalámg
)1
)
Yield
(%)
Puri®cation
(fold)
Crude extract 120.0 1800 ND ND ND ND
Q-Sepharose FF 67.0 101 4.197 41.6 100 1.0
(NH
4
)
2
SO
4
60% 2.5 88 4.047 46.0 96 1.1
Superdex 75 HR 8.0 28 1.890 67.5 45 1.6
MonoQ 4.0 4 1.875 468.8 45 11.3
Phenylsuperose 2.0 1.3 0.750 577.0 18 50.0
Ó FEBS 2002 Poly b-lysine assembly in S. noursei (Eur. J. Biochem. 269) 351
could possibly lie in the same region of the S. noursei
chromosome as the resistance gene. Chromosome w alking

(by subcloning various BamHI or NotI fragments into
pBlueScript and subsequent sequencing) on the overlapping
region of the DNA inserts of phP41 and phN6 revealed
several ORFs in t he 3¢ region of na t1 (Fig. 3). One ORF of
1518 bp was interesting because it encoded a hypothetical
protein of 506 residues with a deduced M
r
of 53 kDa, which
is in the range of the estimated molecular mass of NpsA.
The deduced N-terminal sequence of this enzyme is identical
to the sequence determined by microsequencing of NpsA
(see above), which supports that this gene (designated npsA)
is the gene e ncoding the b-lysine a ctivating enzyme. NpsA
has a typical Streptomyces codon usage with a strong bias
for high G/C content (> 90%) in the third codon positions
of the gene. The overall G/C content of the gene is 75%.
Analysis of the deduced amino-acid sequence of NpsA
revealed high similarity with the a denylation domains of
various NRPSs. NpsA possesses all the 10 conserved
signature sequences A1 to A10 characteristic of the adeny-
lation domains of NRPS [15]. Remarkably, the enzyme h as
95% amino-acid sequence identity with SttA from the
biosynthesis gene cluster of streptothricin in S. rochei,which
con®rms the previously proposed role of the enzyme as a
b-lysine activating enzyme [18].
An alignment of the amino-acid residues of the adenylate
binding pocket of NspA as well as of S ttA with consensus
sequences derived f rom the relevant binding residues in t he
amino-acid bindingpocket of thephenylalanine A-domainof
gramicidin S synthetase [32,33] showed their similarity to the

binding pockets of A-domains, which are known to activate
positively charged amino a cids such as ornithine and lysine.
In particular the characteristic Asp in position 239 of NpsA
indicated a strong relationship t o the lysine-activating
Table 2. Speci®city determining residues in the binding pocket of Np sA. Alignment of the speci®city determining residues of NpsA adenylation
domain with several adenylation domains activating amino acids with positively charged side chains. NpsA (b-lysine, this work), SttA (adenylating
enzyme from S. rochei [27]), BacB_M1 (a-lysine, module 1 of bacitracin synthetase B, accession no. AAC06347), BacB_M2 (a-ornithine, module 2
of bacitracin synthetase B, Acc.No. AAC06347), GrsB_M3 (a-ornithine, module 3 of gramicidin synthetase B, accession no. CAA43838). The
speci®city conferring residues we re aligned according to th e method of S tach elhaus et al. [28]
Enzyme Amino acid
Position in amino-acid binding pocket
234 235 236 239 278 299 301 322 330 331
NpsA b-Lys G D T E G V G T L V
SttA b-Lys G D T E G V G T L V
BacB_M1 a-Lys F D A E S I G S V C
BacB_M2 a-Orn F D V G E I G S V D
GrsB_M3 a-Orn F D V G E I G S L I
Fig. 3.
11
Map of the sequenced region containing the n ourseothricin resistance gene nat1 and the gene npsA encoding the b-lysine activating e nzyme
NpsA. (A) The region represents the overlapping region of phages phP41 and phN6. Arrowheads indicate the orientation and relative length of the
sequenced orfs. Based on the similarities of their deduced amino-acid sequences with proteins in the database, ORFs A±E are proposed to encode
proteins with the following functions: ORF A (acylase), ORF B (thioesterase), ORF C (phosphotransferase), ORF D (unknown), ORF E
(regulator). The same orfs in a similar arrangement have be en shown t o be p resent in a region of the streptothricin b iosynthesis gene c luster of
S. rochei [27] (B) The strategy of expression cloning of npsA isshowninthelowerpartBofthe®gure.TheEcoRI fragment was s ubcloned in
pBluescript and cloned as a Xba I±HindIII fragment into plasmid pDW5 as described in the text. Vegp40 denotes the position of the veg promoter of
B. subtilis [25].
352 N. Grammel et al. (Eur. J. Biochem. 269) Ó FEBS 2002
module of b acitracin synthetase ( Table 2). Interestingly, the
universally conserved Phe234 of NRPS A-domains is

changed into a glycine residue in both NspA and SttA, w hich
may result i n a change of conformation of the Asp235 side
chain possibly binding the unusual b-amino-acid substrate.
Expression of npsA in
S. lividans
To con®rm the identity of the gene npsA as the gene
encoding NpsA, a 3-kb EcoRI fragment from the phage
clone ph41 (Fig. 3) encompassing the entire npsA ge ne was
ligated to EcoRI-cleaved Bluescript vector and after excision
as a HindIII±XbaI fragment was ligated into HindIII±XbaI-
cleaved pDW5, a derivative of p WHM4, under t he control
of the veg promoter from Bacillus subtilis [34] (Fig. 3).
Transformation of S. lividans by the plasmid containing the
cloned gene resulted in strain S. lividans W5. Crude extracts
of strain W5 were prepared as for S. noursei and fraction-
ated on Q-Sepharose FF. Testing the fractions clearly
revealed the presence of the b-lysine activating activity of
NpsA in this S. lividans strain, which was missing in a
control strain containing plasmid pDW5 (not shown).
These data unambiguously indicate that the npsA gene
encodes the b-lysine activating enzyme NpsA.
Detection and puri®cation of a b-lysine binding
protein in
S. noursei
A prerequisite in peptide bond formation between amino
acids in nonribosomal systems is the covalent activation of
amino-acid residues as enzyme-linked t hioesters [12,13]. As
the b-lysine activating enzyme is a stand-alone adenylation
domain lacking a PCP-domain, we set out to identify the
missing amino acyl o r p eptidyl carrier protein which would

represent the rest of the missing part of the putative b-lysine
module.
Using radiolabelled b-lysine as substrate, we tested
fractions of protein extracts from S. noursei for the presence
of a protein that would bind b-lysine covalently after its
activation as adenylate by NpsA. Separation of a crude
extract of S. noursei on a Q Sepharose FF and testing
fractions for covalent binding of radioactive b-lysine in t he
presence of NpsA and ATP revealed a peak of b-ly sine-
binding activity. Gel ®ltration on a Superdex 75 pg column
revealed that the b inding protein has a s urprisingly high M
r
( 70 000 Da) which in view of the small sizes of P CP-
domains ( 100 residues) of NRPS indicates that this
protein must be a multimer or must harbour additional
functions besides binding b-lysine as a thioester. The
b-lysine binding protein eluted independently of the b-lysine
activating enzyme from the Q Sepharose FF column which
indicates that these two enzymes do not form a stable
complex w ith e ach other (not sho wn). To test the nature of
the covalent bond between the binding protein and b-lysine,
the b-lysine binding protein was charged with radioactive
b-lysine and the covalent enzyme±substrate complex was
subjectedtoperformicacid
5
oxidation. The radioactive
b-lysine released from the enzyme was identi®ed by thin
layer chromatography (using solvent systems I and II).
Treatment of charged protein with f ormic acid released no
b-lysine indicating that b-lysine is indeed bound to the

protein as a thioester. Analysis by SDS/PAGE at each stage
of puri®cation (see Materials and Methods)
6
of the b-lysine
binding protein revealed enrichment of a prominent band of
 70 kDa (Fig. 4A). To identify this band as the b-lysine
binding protein, the native enzyme was loaded with
radioactive b-lysine in the presence of NpsA and ATP and
subjected to SDS/PAGE. Figure 4 shows that the 70-kDa
band was speci®cally labeled w ith b-lysine. This reaction
was ATP-dependent and also dependent on the presence of
the b-lysine activating enzyme NpsA. The protein was
named NpsB. Attempts to demonstrate the formation of
b-lysyl±b-lysine or poly ( b-lysine) in incubations containing
puri®edNpsAandNpsBwithATPand[
3
H]b-lysine failed.
No evidence for the formation of s uch products was obtai-
ned either as free or enzyme bound material. Moreover,
Fig. 4. Covalent labelling of NpsB with r adio-
active b-lysine. Partial p uri®ed NpsB (phenyl-
Superose step, see Materials a nd methods) w as
incubated with NpsA, radioactive b-lysine and
ATP. The reaction mixtures were incubated at
28 °C for 30 min 2 mL 5% trichloroacetic
acid was added and precipitated p rote in was
recovered by centrifugation. Protein was sub-
jected to SDS/PAGE (10% SD S/polycaryla-
mide slab). The gel was s ubject ed to
autoradio¯uorography using Amp lify solu-

tion (Amersham) according to the manufac-
turer's instructions. Auto¯uorography was for
6 weeks. The co mplete assay mixture con-
tained 0.1 m
M
b-lysine, 0.1 lCi [
3
H]b-lysine,
18 m
M
ATP, 33 m
M
MgCl
2,
1 pkatal NpsA
and 5 lL phenylsuperose f raction of NpsB
(lane A); lane B with om ission of N psA, lane C
with omission of ATP, lane D with o mission
of NpsB. Left panel: Coomassie B lue- stained
gel. Right panel: Auto¯uorograph of same gel.
Ó FEBS 2002 Poly b-lysine assembly in S. noursei (Eur. J. Biochem. 269) 353
incubation of NpsA and NpsB with ATP, [
3
H]b-lysine a nd
streptolydine-gulosamine did not lead to detectable synthe-
sis of a new compound dependent on streptolydine/gulo-
samine. By contrast, the thin layer chromatograms of
reaction mixtures showed only formation of a free com-
pound during these incubation with an Rf value much
higher than that of b-lysine. The formation of this

compound was ATP-dependent and strictly dependent on
the presence of either NpsA or NpsB. The possibility t hat
this compound must be the spontaneous cyclization product
of the b-lysine thioester (cyclo-b-lysine) could not be
determined due to the lack of authentic reference m aterial.
Cloning of the gene of the b-lysine binding protein
Total protein from the last puri®cation step of NpsB was
subjected to preparative SDS/PAGE and the band repre-
senting the b-lysine binding protein w as in-gel digested with
trypsin. After HPLC separation, six tryptic peptides were
sequenced. Each of the resultant sequences was used to
design PCR primers for both strands. The primers were
used in PCR in all possible combinations to amplify the
gene using chromosomal DNA of S. noursei as template.
One primer pair (see Materials and methods) yielded a PCR
product with seq uences corresponding to the amino-acid
sequence of the binding protein. This indicates that the clone
represents a partial sequence of the gene of the b-lysine
binding protein. The PCR fragment was in turn used as a
probe in plaque hybridization screening of our S. noursei
phage library. From one hybridizing phage, P h1-2, a 15-kb
insert was obtained (not shown). Narrowing down the
hybridizing region by restriction mapping and Southern
analysis led to the subcloning of a 6.8-kb BamHI fragment,
which was partially sequenced. Analysis of the sequence
revealed three ORFs as shown i n Fig. 5. The central gene
encoded a protein with 606 amino acids of a calculated
M
r
=64 100, which contained all of the six internal

sequences obtained from microsequencing of the peptide
fragments, thus con®rming that the gene is npsB.Analysisof
the deduced amino-acid sequence indicated that the protein
is composed of two distinct domains. The ®rst domain
located between amino-acid residues 40±120 has similarity
with various ACP- and PCP-domains of polyketide s ynth-
ases and peptide synthetases. The invariant serine residue
representing the 4¢-phosphopanthetheine attachment site is
located at amino-acid position 88. The second domain of
NpsB located carboxyterminal to t he ACP-domain from
amino-acid residues 150±550 has similarity with condensa-
tion and epimerization domains of a number of peptide
synthetases (Fig. 5). In particular, the sequence from amino-
acid residues 286±296 (HQLAFDMVS) is r eminiscent of
the signature sequences C3 (HHxISDGxS) or E2 (his-motif)
(HHxxxDxVSWxIL) of the C-domains and E-domains of
various peptide synthetases, respectively [15]. Moreover, in a
part of the protein C-terminal to the H-H-motif, all of the
®ve conserved motifs E3 to E7 (in the nomenclature of Konz
& Marahiel [15]) of E-domains are present. Interestingly, the
second His i n the C3 or E2 motifs, which was shown to be
critical both for the condensation of amino acids in
C-domains [35] and the epimerization of amino acids in
E-domains [36] in NpsB, is replaced by glutamine suggesting
a natural mutation from His to Gln in that sequence. As the
con®guration of the b-lysine residues in nourseothricin is
L
and the b-lysine substrate used here was also in the
L
con®guration (obtained by acid hydrolysis of n ourseo-

thricin), the function of this epimerization domain is
dif®cult to discern. Also, it is not known whether
H-Q-versions of the E2/C3 signature sequences are func-
tionally active. Sequencing of the upstream region of the
npsB revealed an ORF encoding a carbamoyltransferase
and further u pstream an IS-like sequence. Th e vicinity of a
gene encoding a carbamoyltransferase directly relates to the
carbamoyl group at the C-5 hydroxyl group of the
gulosamine of nourseothricin and is a further hint that
the cloned region of the S. noursei chromosome with the
b-lysine binding protein gene and the carbamoyltransferase
Fig. 5. Map o f a 6.8-kB Bam HI fragment from
the S. noursei chromosome carrying t he gene
npsB encoding the b-lysine binding protein
NpsB. The s equenc ed region spans from the
indicated (asterisk) Sal ItotheBamHI site on
the left border o f the fragment. Arrows indi-
cate the orientation an d relative l ength of t he
sequenced orfs. Sequencing revealed the g ene
npsB (identi®ed by comparison with sequences
of tryptic peptides derived from NpsB), an orf
(orf1) with a de du ced amino-acid s eq uence
showing homology to carbamoyltransferases,
and a IS-like sequence. In the lower part of the
®gure is shown sch ematically the s tructure of
the NpsB protein with the two-domain
arrangement consisting of a PCP-domain and
a domain with s imilarity to E-domains of
NRPSs.
354 N. Grammel et al. (Eur. J. Biochem. 269) Ó FEBS 2002

gene are part of the nourseothricin biosynthetic gene
cluster.
It is noteworthy, that no overlapping clones c onnecting t he
inserts of phage ph41 and Ph1-2 were found. In fact, the two
inserts appear to be located at a distance of more than
20 kb on the chromosome of S. noursei suggesting a separa-
tion of the b iosynthetic genes in two p artial gene clusters.
DISCUSSION
Streptothricins are nucleoside peptides t hat c ontain a poly
b-lysyl chain which may vary from three to seven residues in
length and which is attached to the 2-amino group of the
gulosamine moiety of the antibiotic via an amide linkage. As
yet, a total cell-free synthesis of nourseothricins has not been
accomplished, nor have partial enzyme activities from
S. noursei been characterized. The poly(b-lysine) chain i s a
unique example of a poly amino-acid chain in a secondary
metabolite. A number of poly(amino acids) such as folyl-
poly c-glutamate play roles in the housekeeping functions of
their producer cells and like the muramyl peptide chains, the
tripeptide glutathione or the
D
-Ala-
D
-Ala dipeptides of the
bacterial cell walls are formed by a mino-acid ligases such as
UDP-N-acetyl-muramoyl-
L
-alanine-glutamate ligase, gluta-
thione synthetase or
D

-Ala-
D
-Ala ligase [37]. They condense
carboxylate-containing compounds via the intermediacy of
acylphosphates as in the case of formation of the
D
-Ala-
D
-Ala dipeptide [38]. Remarkably, in several compounds
synthesized by this mechanism, unusual peptide bonds sych
as the c-glutamyl peptide bond also occur as in the muramyl
peptides and glutathione. As in the p oly(b-lysyl) chains, the
peptide bond is also unusual (x-b-lysyl) and no poly(amino-
acid) synthetases operating via the thiol template mecha-
nism have been described as yet, but it could not be excluded
a priori that poly b-lysyl synthesis would occur via a ligase-
like mechanism.
7
The data presented here, however, show that the poly-
(b-lysine) chain of nourseothricin must be synthesized by
an NRPS-like system in a mechanism that uses NspA, a
stand-alone b-lysine adenylating enzyme (A-domain) and
NspB, a b-lysine binding protein consisting of a PCP-
domain and a domain with similarity to E-domains of
peptide synthetases. N spA i s unique because of its
extraordinarily exclusive substrate speci®city for b-lysine,
which contrasts th e relaxed speci®city of most NRPS. The
sequence of NspA is almost identical with that of SttA the
enzyme which has been shown previously to be involved in
the biosynthesis of streptothricins in S. noursei [18]. The

speci®city-conferring residues of the substrate binding
pocket of NspA (and SstA) display a sequence with
similarity to that of domains known to activate ornithine,
diaminobutyric acid, hydroxyornithine or l ysine but with a
substantial change of the highly conserved Phe234 present
in all NRPS A-domains into a glycine which may lead to
alteration of the overall conformation ofthe active site
pocket which may lead to t he selective binding of b-lysine
to this pocket (Table 2).
For the condensation of the b-lysine residues, covalent
attachment to a PCP-domain in thioester linkage appears to
be necessary similarly to other NRPS systems. Accordingly,
NspA ef®ciently loads NspB with b-lysine in thioester
linkage. S tand-alone adenylation domains in nonribosomal
peptide synthesis have been described for various biosyn-
thesis systems, such as of the aryl peptide lactones, the aryl-
siderophore peptides or in the case of
D
-alanyl-lipoteichoic
acid [39±43
8
], where they activate aromatic carboxylic acids
or an amino acid such as alanine as adenylates
9
,whichin
turn are loaded to speci®c PCP domains. These PCP-
domains are e ither alone-standing PCPs, as i n the b iosyn-
thesis of actinomycin [39] and
D
-alanyl-lipoteichoic a cid, or

they are fused to protein domains catalysing another step of
the same pathway as in EntB, the PCP for the 2,3-
dihydroxybenzoic acid in enterobactin synthesis [40]. Usu-
ally, the PCP-bound carboxylates are condensed with the
next amino acids in the biosynthetic sequence by the action
of C-domains or Cy-domains forming part of the down-
stream module. Cy-domains not only catalyse condensa-
tions between serine, threonine or cysteine residues with
upstream thioester-activated amino a cids but after conden-
sation also cyclize their substrate to the corresponding
oxazoline and thiazoline, respectively [44].
In contrast to these examples, the b-lysine-binding PCP-
domain described here is fused to a domain w ith similarity
to the E-domains of NRPS, which suggests a different
mechanism. E-domains in NRPS are always located
downstream of PCP-domains and catalyse the conversion
of the amino acid or peptidyl intermediate tethered to the
PCP-domain from the
L
to the
D
con®guration. In NRPSs
which do not epimerize their substrates, C-domains are also
always directly located downstream of PCP-domains. The
mechanistic basis for both E-domains and C-domains is
similar. They both contain a double His-motif (E2 and C3,
respectively) f rom w hich the second His of the E-domain is
postulated to remove a proton either from the a-carbon of
the thioester-activated amino acid or peptidyl intermediate
leaving a carbanion intermediate ready for attack by a

proton in an S
n
2 mech anism [44]. In the C3 signature
sequence of C-domains, the second His is thought to remove
a proton f rom the amino nitrogen of the acceptor amino-
acid in the condensation p rocess [36]. Thus, the signi®cance
of the E-domain described here is not clear because the
second His in the E2 is missing the motif being changed into
a H-Q, which would suggest that the E-domain of NspB
would be nonfunctional. On the other hand, Cy-domains
that are present in NRPS catalysing the condensation of a
serine, threonine or cysteine residue with an upstream
residue under subsequent heterocycle formation have a
modi®ed His-motif with only one H which leaves the
possibility open for a specialized function of the modi®ed
E-domain d escribed here. PksF [45] and PpsE [46] encoded
by genes from the genomes of B. subtilis and M. tubercu-
losis, respectively, are other examples of E-domains in which
the second His of the E2-sequence is changed into Q and A,
respectively. It h as not yet been tested whether these
E-domains function as E- or C-domains. Moreover, the
modi®ed E 2 motif suggests s ome signi®cance in the light of
the fact that in NpsB an epimerization by the established
mechanism [36] of proton abstraction from a-carbon would
remain undetectable, because the a-carbon of b-lysine is not
asymmetric. T hus, if the E-domain of NpsB would have a
function in nourseothricin biosynthesis, it could be specu-
lated th at t his e nzyme might catalyse b-lysine condensation
in conjunction either with a C-domain or condensing
enzyme. As yet, we have not been able to demonstrate the

formation of a b-lysyl-b-lysine with NpsA and NpsB nor the
transfer of b-lysine to gulosamine, which rules out a direct
Ó FEBS 2002 Poly b-lysine assembly in S. noursei (Eur. J. Biochem. 269) 355
function of the E-domain of NspB as a condensing domain.
Interestingly, Stachelhaus et al. reported the involvement of
the E-domain of GrsA (PheATE) in conjunction with the
ProCAT module of GrsB in the condensation of phenyl-
alanine with proline [35]. This transfer role was independent
from the presence or absence of the second His in the
E2-motif of that E-domain. Thus, one has to consider the
possibility of an additional factor operating in the conden-
sation reaction between the b-lysine residues of the poly-
(b-lysine) chain as well as the condensation reaction of
carboxyl activated b-lysine with the gulosamine moiety. The
fact, that these condensation reactions involve the partici-
pation of only one single stand-alone A-domain and a single
binding protein NspB points to an iteration of these two
proteins in the a ssembly line of poly b-lysine chains in
nourseothricin biosynthesis. This cou ld either take place by
the formation of a multimeric complex between the two
enzymes or by an iterative mechanism of poly b-lysine chain
formation on a single NspA/NspB module. To address
these questions as well as the t rue function of the E-domain
of NpsB in the condensation of b-lysine r esidues with each
other and also with the gulosamine moiety of nourseothri-
cin, future studies of nourseothricin biosynthesis and t he
cloning and sequencing of more genes in the cluster and
their expression as functional active enzymes will be
necessary.
ACKNOWLEDGEMENTS

We thank Prof. Perry A . Frey and Prof. Udo Grae fe for providing us
with b-ly sine, Michael Gru
È
n and Kerstin Webe r for skilled tec hnical
assistance and Prof. Albert Hinnen for his support of this work. We
also thank Prof. Jerald C. Ensign and Dr Sandor Biro
Â
for critical
reading of the manuscript.
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