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 involvedin poly b-lysine chain assembly in nourseothricin synthesis Nicolas Grammel1,*, Kvitka Pankevych2, Julia Demydchuk2,

A b-lysine adenylating enzyme and a b-lysine binding protein involved

in poly b-lysine chain assembly in nourseothricin synthesis

Nicolas Grammel1,*, Kvitka Pankevych2, Julia Demydchuk2, Klaus Lambrecht2, Hans-Peter Saluz2,

Ullrich Keller1and Hans KruÈgel2

1 Max-Volmer-Institut fuÈr Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie,

Technische UniversitaÈt Berlin, Germany; 2 Department of Cell and Molecular Biology, Hans KnoÈll 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 Mr56 000 that speci®cally activates b-lysine by

adenylation but does not bind to it as a thioester Cloning

and sequencing of npsA from S noursei including its

¯ank-ing DNA regions revealed that it is closely linked to 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 Mr

64 100 which can be loaded by NpsA with b-lysine as a thioester Analysis of the deduced amino-acid sequence from the gene (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 either the domain is nonfunctional or has

a specialized function The presence of 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 chain

Keywords: nonribosomal peptide synthetase; PCP-domain, b-lysine, nourseothricin, Streptomyces

The nourseothricins belong to the family of the

streptothri-cin antibiotics that are produced by various streptomycete

strains such as Streptomyces noursei [1] These compounds

are nucleoside peptides 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, streptothricins are also being tested as fungistatics in agriculture for the treatment of blast disease and other plant diseases [7] Knowledge of the biosynthesis of streptothricins mainly stems from 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 features of peptides and nucleosides, their biosyn-thesis involves quite diverse enzyme activities for sugar biosynthesis, peptide 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 KruÈgel, Department of Cell and Molecular

Biology, Hans KnoÈll Institute for Natural Product Research,

Beutenbergstrảe 11, D-07745 Jena, Germany,

Fax: + 49 3641 656694, Tel.: + 49 3641 656684,

E-mail: hkruegel@pmail.hki-jena.de, or U Keller,

Max-Volmer-Institut fuÈr Biophysikalische Chemie und

Biochemie, Fachgebiet Biochemie und Molekulare Biologie,

Techni-sche UniversitaÈt Berlin, Franklinstrasse 29, D-10587 Berlin, Germany.

Tel.: + 49 30 314 25653, E-mail: Ullrich.Keller@TU-Berlin.de

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: ActinoDrug Pharmaceuticals GmbH, Hennigsdorf,

Germany.

(Received 7 June 2001, revised 11 October 2001, accepted 6 November

2001)

amino acid into a given peptide product [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 binding 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 such as

folyl-poly c-glutamate, an amino-acid folyl-polymer produced by

bacteria and eukaryotes [16], poly 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 disruption 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 from 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 synthesized by a thiol template mechanism

M A T E R I A L S A N D M E T H O D S

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 medium M79 [20] Mycelia for enzyme preparations were harvested from cultures propagated in 500 mL conical ¯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 of nourseothricin and its biosynthetic precursors.

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 [3H]b-Lysine (220 Ciámmol)1) was

obtained from Hartmann Analytik, GMbH, Braunschweig,

Germany

2 [32P]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 mMMgCl2

for 1 h After centrifugation at 30 600 g

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 eluted with a 200-mL

linear gradient from 0 to 0.2M 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% The solution was then left on ice for at least 2 h The

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

1 mLámin)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 0.2M NaCl in buffer B (¯ow rate 1 mLámin)1, gradient

60 min) Fractions containing enzyme activity were pooled and saturated ammonium sulphate solution was added 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 (PharmaPharma-cia) The ¯ow rate was 300 lLámin)1and 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 cold room S noursei mycelia (40 g) suspended in 200 mL of buffer B was passed through a French pressure cell at 68 947 kPa DNAse I (grade II, Sigma) was added at 50 lgáL)1 and MgCl2 at

20 mM and left on ice with stirring for 1 h After centri-fugation for 20 min at 30 600 g

adjusted to a conductivity of 4 mS with water (containing

10 mMdithioerythritol) 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±0.2M

Fig 2 Identi®cation of NpsA by gel ®ltration

of b-lysine activating enzyme on Superose 12.

Concentrated enzyme (300 lL) from the

phenlysuperose step of Table 1 were applied

onto a Superose 12 column (Pharmacia).

300 lL fractions were collected The

frac-tionation range from fraction 35±55 is shown.

(3/4) Absorbance at 280 nm; (bars) activity

pattern of the b-lysine-dependent ATP/PP i

exchange The inset shows SDS/PAGE (10%

polyacrylamide, according to [22]) of 30 lL

portions of the indicated fractions Staining

was with Coomassie blue The band

repre-senting the b-lysine activating enzyme NpsA is

denoted by an arrow.

NaCl was passed through the column and 5-mL fractions

were collected

Fractions were assayed by determination of covalent

binding of [3H]b-lysine to protein in the presence of the

b-lysine activating enzyme, ATP and MgCl2 (see below)

Fractions containing b-lysine binding activity were pooled

and saturated ammonium sulphate solution was added until

70% saturation After leaving on ice overnight, the

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 column,

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) ranging 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 mMb-lysine, 2.5 mMATP, 5 mMMgCl2, 0.1 mM

tetrasodium pyrophosphate and 2 ´ 105c.p.m [32

P]tetra-sodium pyrophosphate and 10±50 lL of b-lysine activating

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 mM

b-lysine, 0.1 lCi [3H]b-lysine, 18 mMATP, 33 mM MgCl2

and 5±25 lL of b-lysine binding protein fraction in a total

volume of 60 lL After 30 min of incubation at 28 °C,

2 mL 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 0.1MTris/HCl, pH 8.0, 4 mM dithio-erythritol, 1 mMbenzamidine, 1 mMphenylmethylsulfonyl

¯uoride, 2 mMEDTA Buffer C was the same as buffer B except that it contained 0.05MTris/HCl, pH 7.5 Solvent systems for thin layer chromatography of b-lysine were n-butanol/aceticacid/water(4 : 1 : 1,v/v/v;solventsystem 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 scintillascintilla-tion 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 thioester 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 trypsin 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 plaques with the nourseothricin resistance gene nat1 [4±6], and 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

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)

R E S U L T S

b-Lysine activation inS noursei

Peptide synthetases activate their amino-acid substrates by

adenylation with 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 to 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 correlated with the intensity of a particular band of 56 kDa (Fig 2) The enzyme was named NpsA (nourseothricin peptide synthetase A) Microsequencing of this band yielded the N-terminal sequence: MESS ASSFLEPFFDVXR

Characterization of the b-lysine activating enzyme (NpsA) fromS noursei

Passing NpsA through a calibrated Superdex 75 pg gel

®ltration column revealed that the enzyme has an Mr between 58 000 and 60 000 This ®tted with the estimated molecular mass of 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 MgCl2did 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-arginine not tested) This indicates that the active site of the enzyme can strictly distinguish between an a-amino and a b-amino group of lysine Other 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 part of the nourseothricin synthesizing enzyme system

No other b-lysine activating enzymes were detected in

S noursei

Cloning of theNpsA gene Phages ph41 and phN6 are overlapping clones obtained from screening of 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 activating enzyme

Table 1 Puri®cation of the b-lysine activating enzyme NpsA from Streptomyces noursei Cells (50g) from a 72-h culture of S noursei was used Puri®cation was based on ATP-pyrophosphate exchange dependent on the presence of b-lysine One nkatalámol )1 is the amount of enzyme catalysing the exchange of 1 nmol of pyrophosphate into ATP per second ND, not determined.

Puri®cation

step Volume(mL) Protein(mg) Activity(nkatal) Speci®c activity(pkatalámg )1 ) Yield(%) Puri®cation(fold)

could possibly lie in the same region of the S noursei

chromosome as the resistance gene Chromosome walking

(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 the 3¢ region of nat1 (Fig 3) One ORF of

1518 bp was interesting because it encoded a hypothetical

protein of 506 residues with a deduced Mrof 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 encoding the b-lysine activating 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 adenylation 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 has 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 SttA with consensus sequences derived from the relevant binding residues in the amino-acidbindingpocketofthephenylalanineA-domainof gramicidin S synthetase [32,33] showed their similarity to the binding pockets of A-domains, which are known to activate positively charged amino acids such as ornithine and lysine

In particular the characteristic Asp in position 239 of NpsA indicated a strong relationship to the lysine-activating

Table 2 Speci®city determining residues in the binding pocket of NpsA 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 were aligned according to the method of Stachelhaus et al [28]

Enzyme Amino acid

Position in amino-acid binding pocket

Fig 3.

11 Map of the sequenced region containing the nourseothricin resistance gene nat1 and the gene npsA encoding the b-lysine activating enzyme 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 been shown to be present in a region of the streptothricin biosynthesis gene cluster of

S rochei [27] (B) The strategy of expression cloning of npsA is shown in the lower part B of the ®gure The EcoRI fragment was subcloned in pBluescript and cloned as a XbaI±HindIII fragment into plasmid pDW5 as described in the text Vegp40 denotes the position of the veg promoter of

B subtilis [25].

module of bacitracin synthetase (Table 2) Interestingly, the

universally conserved Phe234 of NRPS A-domains is

changed into a glycine residue in both NspA and SttA, which

may result in a change of conformation of the Asp235 side

chain possibly binding the unusual b-amino-acid substrate

Expression of npsA inS 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 gene 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 pWHM4, under the 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 inS noursei

A prerequisite in peptide bond formation between amino

acids in nonribosomal systems is the covalent activation of

amino-acid residues as enzyme-linked thioesters [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 or peptidyl 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 the presence of NpsA and ATP revealed a peak of b-lysine-binding activity Gel ®ltration on a Superdex 75 pg column revealed that the binding protein has a surprisingly high Mr ( 70 000 Da) which in view of the small sizes of PCP-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 with each other (not shown) 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 subjected to performic acid

b-lysine released from the enzyme was identi®ed by thin layer chromatography (using solvent systems I and II) Treatment of charged protein with formic 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)

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 with 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®ed NpsA and NpsB with ATP and [3H]b-lysine failed

No evidence for the formation of such products was obtai-ned either as free or enzyme bound material Moreover,

Fig 4 Covalent labelling of NpsB with

radio-active b-lysine Partial puri®ed NpsB

(phenyl-Superose step, see Materials and methods) was

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 protein was

recovered by centrifugation Protein was

sub-jected to SDS/PAGE (10%

SDS/polycaryla-mide slab) The gel was subjected to

autoradio¯uorography using Amplify

solu-tion (Amersham) according to the

manufac-turer's instructions Auto¯uorography was for

6 weeks The complete 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 fraction of NpsB

(lane A); lane B with omission of NpsA, lane C

with omission of ATP, lane D with omission

of NpsB Left panel: Coomassie Blue-stained

gel Right panel: Auto¯uorograph of same gel.

incubation of NpsA and NpsB with ATP, [3H]b-lysine and

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 that

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 material

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 was 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 sequences 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, Ph1-2, 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 in Fig 5 The central gene

encoded a protein with 606 amino acids of a calculated

Mr=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 Analysis of 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 synth-ases and peptide synthetsynth-ases 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 the ACP-domain from amino-acid residues 150±550 has similarity with condensa-tion and epimerizacondensa-tion domains of a number of peptide synthetases (Fig 5) In particular, the sequence from amino-acid residues 286±296 (HQLAFDMVS) is reminiscent 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 in 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

Lcon®guration (obtained by acid hydrolysis of nourseo-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 upstream an IS-like sequence The 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 of a 6.8-kB BamHI fragment from the S noursei chromosome carrying the gene npsB encoding the b-lysine binding protein NpsB The sequenced region spans from the indicated (asterisk) SalI to the BamHI site on the left border of the fragment Arrows indi-cate the orientation and relative length of the sequenced orfs Sequencing revealed the gene npsB (identi®ed by comparison with sequences

of tryptic peptides derived from NpsB), an orf (orf1) with a deduced amino-acid sequence showing homology to carbamoyltransferases, and a IS-like sequence In the lower part of the

®gure is shown schematically the structure of the NpsB protein with the two-domain arrangement consisting of a PCP-domain and

a domain with similarity to E-domains of NRPSs.

gene are part of the nourseothricin biosynthetic gene

cluster

Itisnoteworthy, thatnooverlappingclones connectingthe

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 biosynthetic genes in two partial gene clusters

D I S C U S S I O N

Streptothricins are nucleoside peptides that contain 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 is 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 theD-Ala-D-Ala dipeptides of the

bacterial cell walls are formed by amino-acid ligases such as

UDP-N-acetyl-muramoyl-L-alanine-glutamate ligase,

gluta-thione synthetase orD-Ala-D-Ala ligase [37] They condense

carboxylate-containing compounds via the intermediacy of

acylphosphates as in the case of formation of theD

-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 poly(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 PCP-domain with similarity to E-PCP-domains of

peptide synthetases NspA is unique because of its

extraordinarily exclusive substrate speci®city for b-lysine,

which contrasts the 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 lysine 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 the 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 Stand-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 ofD-alanyl-lipoteichoic acid [39±43

8 ], where they activate aromatic carboxylic acids

or an amino acid such as alanine as adenylates

turn are loaded to speci®c PCP domains These PCP-domains are either alone-standing PCPs, as in the biosyn-thesis of actinomycin [39] andD-alanyl-lipoteichoic acid, 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 acids 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 PCP-domain with 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 theLto theDcon®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) from which 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 Sn2 mechanism [44] In the C3 signature sequence of C-domains, the second His is thought to remove

a proton from the amino nitrogen of the acceptor amino-acid in the condensation process [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 described 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 has not yet been tested whether these E-domains function as E- or C-domains Moreover, the modi®ed E2 motif suggests some 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 Thus, if the E-domain of NpsB would have a function in nourseothricin biosynthesis, it could be specu-lated that this enzyme 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

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 assembly line of poly b-lysine chains in

nourseothricin biosynthesis This could 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 true function of the E-domain

of NpsB in the condensation of b-lysine residues with each

other and also with the gulosamine moiety of

nourseothri-cin, future studies of nourseothricin biosynthesis and the

cloning and sequencing of more genes in the cluster and

their expression as functional active enzymes will be

necessary

A C K N O W L E D G E M E N T S

We thank Prof Perry A Frey and Prof Udo Graefe for providing us

with b-lysine, Michael GruÈn and Kerstin Weber for skilled technical

assistance and Prof Albert Hinnen for his support of this work We

also thank Prof Jerald C Ensign and Dr Sandor Biro for critical

reading of the manuscript.

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