1 Type II thioesterase ScoT from 2 3 propionate....

1

Type II thioesterase ScoT from Streptomyces coelicolor A3(2) modular 1

polyketide synthase Cpk hydrolyses acyl residues with preference for 2

propionate. 3

4

Magdalena Kotowska1*, Krzysztof Pawlik

1, Aleksandra Smulczyk-Krawczyszyn

1**, 5

Hubert Bartosz-Bechowski2, Katarzyna Kuczek

1 6

7

1 Department of Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental 8

Therapy, Polish Academy of Sciences, ul. Weigla 12, 53-114 Wroclaw, Poland 9

2 Faculty of Chemistry, University of Wroclaw, ul. F. Joliot-Curie 14, 50-383 Wroclaw, 10

Poland 11

*Corresponding author. Mailing address: Department of Microbiology, Ludwik Hirszfeld 12

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 13

12, 53-114 Wroclaw, Poland. Phone: +48 (71) 3371172 ext. 184. Fax: +48 (71) 3371382. E-14

mail: [email protected] 15

**Present address: MEDianus sp. z o.o., ul. Cystersow 13/2, 31-553 Krakow, Poland 16

17

Running title: 18

Thioesterase ScoT prefers propionate. 19

20

Keywords: 21

Polyketide, Modular polyketide synthase, Type II thioesterase, Streptomyces coelicolor 22

23

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01371-08 AEM Accepts, published online ahead of print on 12 December 2008

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Abbreviations: 1

ACP: acyl carrier protein, DEBS: 6-deoxyerythronolide B synthase, FAS: fatty acid synthase, 2

NAC: N-acetylcysteamine, NRPS: nonribosomal peptide synthetase, PCP: peptidyl carrier 3

protein, PKS: polyketide synthase, p-NP: p-nitrophenyl, 4’PP: 4’-phosphopantetheine, TE II: 4

type II thioesterase 5

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ABSTRACT 1

2

Type II thioesterases (TE IIs) were shown to maintain efficiency of polyketide 3

synthases (PKSs) by removing acyl residues blocking extension modules. However, these 4

enzymes differ in their substrate specificity and kinetic parameters, which may have 5

significant consequences when including them in engineered hybrid systems for the 6

production of novel compounds. Here we show that thioesterase ScoT associated with 7

polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyse acetyl, 8

propionyl and butyryl residues which is consistent with its editing function. This enzyme 9

clearly prefers propionate, which is in contrast to TE IIs tested so far and indicates its possible 10

role in control of the starter unit. We also report activity of ScoT mutants and conclude that 11

this enzyme is an α/β hydrolase with Ser90 and His224 in the active site. 12

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INTRODUCTION 1

2

Modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) 3

are complexes of multifunctional enzymes with exceptionally high molecular masses (300-4

1600 kDa), which produce a variety of biologically active compounds. Despite substantial 5

structural differences between the two groups of natural products (polyketides and non-6

ribosomal peptides), the multienzymes responsible for their biosynthesis show important 7

functional analogies (7, 37, 39). Enzymatic domains responsible for one round of chain 8

extension are grouped in modules. During synthesis all substrates and intermediates are 9

covalently bound to the enzyme by a thioester linkage with a 4’-phosphopantetheine (4’PP) 10

arm of an acyl carrier protein (ACP) domain in PKS or peptidyl carrier protein (PCP) domain 11

in NRPS. Full-length chain is usually released by a thioesterase domain located at the C-12

terminus of the last module. Both polyketides and peptides usually undergo further 13

modifications before becoming final biologically active compounds. 14

In bacteria, genes coding main PKS or NRPS subunits are clustered with genes coding 15

enzymes modifying polyketide or peptide chain, as well as regulatory and resistance genes. 16

Many modular PKS and NRPS clusters include also genes for type II thioesterases (TE IIs). 17

These discrete, 25 - 29 kDa proteins were first identified in some mammalian fatty acid 18

synthase (FAS) complexes, where they are alternative chain terminating enzymes with 19

affinity for medium-chain-length fatty acids (38). In PKS and NRPS systems they appear to 20

have an “editing” role – removing non-reactive acyl residues blocking the multienzyme (16, 21

36, 50), removing amino acids that can not be elongated (48) and/or controlling the starter 22

unit selection (20). However, recent reports on polyether ionophore antibiotics biosynthesis 23

show that TE IIs are responsible for the release of this class of polyketides by hydrolysis (15, 24

28, 29). 25

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Biosynthesis of polyketides involves repeated cycles of condensation and reduction of 1

short carboxylic acids in a manner similar to fatty acid biosynthesis (39). The first step of 2

each biosynthetic cycle is a decarboxylative condensation of a starter unit (or the growing 3

chain from the previous module) with a malonyl-CoA derived extender unit. This results in 4

chain elongation by a dicarbon unit and generates a β-carbonyl group which can be further 5

reduced depending on the presence of reducing domains in a given module. The chain is then 6

transferred to the subsequent module which performs the next elongation cycle. Sometimes, 7

extender units attached to the ACP are decarboxylated without condensation. Products of such 8

aberrant decarboxylation (acetyl, propionyl, butyryl residues) remain covalently attached to 9

the ACP and block the “assembly line”. They must be removed by TE II to restore the PKS 10

activity (16). 11

Since the nonribosomal peptide biosynthesis process does not involve 12

decarboxylation, a different mechanism was proposed to give rise to non-reactive acyl 13

residues (36). After the synthesis of NRPS protein each of the PCP domains is converted into 14

its holo form by attaching 4’-phosphopantetheine in the process called priming catalysed by a 15

4’PP transferase. 4’PP transferases use CoA as a source of 4’PP, but they were also shown to 16

accept different acyl-CoA derivatives. Transfer of acyl-4’PP onto PCP domain is called 17

mispriming and results in inactive NRPS. The role of TE II was proposed to be hydrolysis of 18

these acyl residues and was named deblocking after mispriming (36). The ACP domains of 19

modular PKSs also require priming with 4’PP, therefore it is very likely, that the deblocking 20

after mispriming is also the role of TE II in polyketide synthesis. 21

Biochemical studies show that type II thioesterases are able to hydrolyse acyl residues 22

which may arise from aberrant decarboxylation of extender units or from mispriming of 23

ACP/PCP domains with acyl-4’PP (16, 23, 36, 48, 50). These findings are consistent with 24

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results of disruption experiments, where lack of TE II resulted in drastically reduced levels of 1

polyketide (6, 10, 50) or peptide (11, 35, 49) compounds production. 2

On the contrary, deletion of the TE II gene (pikAV) from picromycin/methymycin 3

synthase gene cluster of Streptomyces venezuelae was found to have no influence on the level 4

of polyketides production (8). This finding is in contrast with an earlier report of Xue et al. 5

(47) who found less than 5% of glycosylated products and no intermediates accumulated by a 6

mutant with disrupted pikAV gene. Therefore the role of PikAV thioesterase remains unclear. 7

Overexpression of pikAV gene in S. venezuelae resulted in 50 – 70% decrease of polyketide 8

production, which can be attributed to hydrolysis of correct extender units (23). On the other 9

hand, polyketide production by picromycin/methymycin synthase expressed in Streptomyces 10

lividans was enhanced up to 7-fold by co expression of PikAV thioesterase (42). 11

Disruption of the TE II gene associated with 6-deoxyerythronolide B synthase (DEBS) 12

of Saccharopolyspora erythraea caused a notable increase of shunt products formed by 13

utilization of acetate instead of propionate as a starter unit, while the combined level of all 14

polyketides produced was reduced by less than 20% (20). DEBS TE II showed in vitro strong 15

preference for the acetyl group bound to the ACP from the loading module of DEBS. 16

Therefore it was proposed that removal of acetate from the loading module ACP is the main 17

function of this TE II (20). 18

This paper deals with a type II thioesterase ScoT associated with a type I modular 19

polyketide synthase Cpk from Streptomyces coelicolor A3(2) (32). ScoT was shown 20

previously to restore polyketide production in a mutant of Streptomyces fradiae lacking its 21

native TE II (tylO from tylosin synthase cluster) (25). It was not possible to study its role in 22

the natural context, since the polyketide produced by the Cpk synthase has not been identified 23

yet. Here we describe kinetic measurements of the activity of thioesterase ScoT towards 24

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synthetic substrates mimicking acyl chains blocking the PKS. We also report activity of ScoT 1

active site mutants. 2

3

MATERIALS AND METHODS 4

5

Materials. 6

N-acetylcysteamine acetate was synthesized by reacting N-acetylcysteamine (NAC) 7

with acetic anhydride. NAC propionate and butyrate were prepared by reacting NAC with 8

appropriate acid in the presence of dicyclohexylcarbodiimide. Solvents were removed in 9

vacuo. Products were purified by chromatography on silica gel column impregnated with 10

anhydrous copper sulphate (13). Obtained compounds were verified by 1H NMR (500 MHz) 11

and ES-MS. 12

p-Nitrophenyl (p-NP) acetate and propionate were obtained from Sigma and p-NP 13

butyrate – from Fluka. Other chemical reagents were from Sigma and Roth. Restriction 14

enzymes and polymerases, as well as DNA and protein mass markers were from MBI 15

Fermentas. 16

E. coli strains were cultured in LB medium (34). 17

18

Construction of the TE II expression plasmids. 19

DNA manipulations were performed according to standard procedures (34). 20

Thioesterase II gene scoT from S. coelicolor A3(2) was amplified by PCR using as a template 21

plasmid pMK15 - a construct in pBluescriptSK(+) containing two BamHI fragments from the 22

T3 terminus of cosmid 1G7 (accession number: AF109727). Primers TE-P-Nde (5’ TTT TTT 23

TTT CAT ATG GGA AGT GAC TGG TT 3’) and TE-K-Hind (5’ TTT TTT TAA GCT TGT 24

CGT ACG TAC ACG GA 3’) were designed to introduce restriction sites (underlined). The 25

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PCR product was cloned in pGEM-T Easy vector (Promega), digested with NdeI and HindIII 1

and cloned into the respective sites of pET-28a(+) to give plasmid pOS22. 2

Mutated versions of ScoT thioesterase carrying mutations S90A and S90C were 3

constructed by overlap extension (34). Upstream and downstream portions of scoT gene were 4

amplified using Pfu polymerase generating blunt ends with appropriate primer pairs 5

introducing mutations (table 1). The upstream part of scoT gene was amplified with TE-P-6

Nde and mutagenic primer marked with “R”, the downstream part of gene – with TE-K-Hind 7

and mutagenic primer marked with “F”. In the subsequent step upstream and downstream 8

parts of scoT gene with mutation (approx. 100 ng each) were mixed and the whole mutated 9

gene was amplified with primers TE-P-Nde and TE-K-Hind to allow cloning in pET-28a(+) 10

digested with NdeI and HindIII. For cloning in pGEX-6P-1, the NdeI and HindIII adhesive 11

ends were filled with Klenow enzyme and the fragment was ligated with SmaI digested 12

vector. 13

Cloned DNA fragments generated by PCR were verified by sequencing. E. coli DH5α 14

strain was used for the cloning steps. Expression plasmids were used to transform E. coli 15

BL21(DE3)pLysS and E. coli BL21Star(DE3) strains. 16

17

Purification of the recombinant proteins. 18

Wild type ScoT protein and ScoT_H224Y mutant was isolated from E. coli 19

BL21(DE3)pLysS cells. Proteins ScoT_S90A and ScoT_S90C were isolated from E. coli 20

BL21Star(DE3) strain. ScoT_H210Y protein was isolated from E. coli BL21Star(DE3) cells 21

additionally transformed with pGroESL plasmid coding chaperones (14). 22

200 ml cultures of E. coli cells transformed with expression plasmids were grown in 23

1 liter flasks in LB medium with appropriate antibiotics (30 µg/ml kanamycin for pET28a(+), 24

100 µg/ml ampicillin for pGEX-6P-1, 34 µg/ml chloramphenicol for pLysS and pGroESL) at 25

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37°C until required OD600 was reached (0.6 for ScoT_H210Y, 1.0 for the remaining proteins). 1

Then the cultures were cooled for 20 min and protein expression was induced with IPTG 2

(isopropyl thio-β-D-galactoside) (0.1 mM for ScoT_H224Y, 0.01 mM for the remaining 3

proteins). After overnight incubation on a shaker (at 22°C for ScoT_H210Y, 9°C for the 4

remaining proteins) the cells were harvested by centrifugation (5000 x g, 10 min, 4°C) and 5

frozen at –70°C. 6

Recombinant proteins with N-terminal His6-tag were isolated by nickel affinity 7

chromatography. Cells were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 8

pH 8.0) (4 ml of lysis buffer/1 g of wet cells) and disrupted by sonication on ice 2 x 5 min 9

(cycle 0.5, amplitude 60%) using the ultrasonic disintegrator UP200S (Dr. Hielscher GmbH). 10

Cell lysate was clarified by centrifugation (10000 x g, 30 min, 4°C). 2 ml of HIS-Select 11

Nickel Affinity Gel (Sigma) previously equilibrated with lysis buffer was added to 50-100 ml 12

of the lysate and allowed to bind for 1 h with gentle shaking at 4°C. The resin was then 13

collected by centrifugation (5000 x g, 5 min, 4°C) and packed into a column. The column was 14

washed with lysis buffer containing 10 mM imidazole and the recombinant protein was eluted 15

with the same buffer containing 50 mM imidazole. Fractions containing the recombinant 16

protein were pooled and desalted using a PD-10 column (GE Healthcare). Buffer was changed 17

to 50 mM Tris-HCl, pH 7.5. Next, 1 ml of Q Sepharose (GE Healthcare) equilibrated with the 18

same buffer was added, allowed to bind for 30 min, packed into a column and washed. The 19

protein of interest was eluted with 100 mM NaCl in 50 mM Tris-HCl, pH 7.5. 20

ScoT_H224Y expressed as a fusion protein with glutathione-S-transferase (GST) was 21

purified by affinity chromatography. Cells collected from 2 l culture were resuspended in 22

100 ml of ATP containing buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgSO4, 2 mM ATP) and 23

disrupted by sonication as described above. 1 ml of Glutathione-Sepharose 4B (GE 24

Healthcare) equilibrated with the same buffer was added to the cleared lysate and allowed to 25

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bind for 30 min. with gentle shaking at 4°C. The resin was then collected by centrifugation 1

(5000 x g, 5 min, 4°C), packed into a column, washed with ATP buffer and equilibrated with 2

PreS buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT). The 3

column bound fusion protein was digested overnight, at 4°C, with 80 U of PreScission 4

protease (GE Healthcare). Purified ScoT_H224Y protein was eluted with PreS buffer. 5

Samples from different purification steps were analysed by SDS-PAGE. Gels were 6

visualized by staining with Coomassie brilliant blue. Protein concentration was determined by 7

the method of Bradford (1976). Fractions containing purified protein were pooled, glycerol 8

was added to the concentration 10% (v/v), aliquots were shock-frozen in liquid nitrogen and 9

stored at –70°C. 10

11

Kinetic assays. 12

Conditions for the enzymatic activity assays were taken from (16). Hydrolysis rates 13

were measured at 30°C in the total volume of 1 ml in the following assay buffer: 200 mM 14

potassium phosphate (pH 7.5), 2.5 mM Tris-HCl (pH 7.5), 50 µM EDTA. Variable amounts 15

of substrates were added from stock solutions in DMSO, and DMSO concentration was 16

adjusted to 3% (v/v). ScoT concentration was 225 nM for p-NP butyrate, NAC butyrate and 17

NAC acetate and 75 nM for the remaining substrates hydrolysis. Concentrations of mutant 18

proteins ScoT_S90C, ScoT_H210Y and ScoT_H224Y were 800 nM, 75 nM and 150 nM, 19

respectively. All samples were measured in triplicate and corrected for background hydrolysis 20

in the absence of the enzyme. The rate of hydrolysis was proportional to the enzyme 21

concentration, therefore to enable comparison of the results on the same plot all data were 22

recalculated to correspond to 75 nM enzyme. 23

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Hydrolysis p-nitrophenyl esters was visible by formation of yellow p-nitrophenolate 1

anion and was followed by spectroscopy (λmax = 400 nm, ε = 8570 M-1

cm-1

). Stock solutions 2

of p-NP esters were 100 mM in DMSO. 3

Hydrolysis of NAC thioesters was followed by inclusion of 0.2 mM DTNB (5,5’-4

dithio-2-nitrobenzoic acid, Ellman’s reagent) in the reaction mixture. The yellow product of 5

the reaction of DTNB with free thiol groups (5-thio-2-nitrobenzoic acid) was monitored by 6

spectroscopy (λmax = 412 nm, ε = 13600 M-1

cm-1

). Stock solutions of NAC thioesters were 7

2 M in DMSO. 8

9

ESI-MS analysis 10

For ESI-MS (Electrospray Ionisation - Mass Spectrometry) experiments the 11

micrOTOF-Q mass spectrometer (Bruker) was used. Buffer for ScoT and ScoT_S90A 12

proteins was changed to 20 mM NH4HCO3 using the PD-10 column (GE Healthacare). 10 µl 13

of protein solution (8.5 µM) was diluted with 300 µl of a solution of acetonitrile/water/formic 14

acid (500:500:1 v/v). In order to determine the presence of acylated thioesterase, 1 µl of 15

substrate solution (10 mM p-NP esters in acetonitrile) was added to 10 µl of protein sample, 16

incubated for 20 seconds at room temperature and diluted with 300 µl of 17

acetonitrile/water/formic acid. Samples were introduced into the ion source at a flow rate of 18

4 µl min-1

. Spectra were scanned from m/z 200 to m/z 2200 and analyzed using the Bruker 19

Daltonics DataAnalysis 3.4 software. The mass scale was calibrated with Tune Mix (Agilent). 20

21

RESULTS 22

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Heterologous expression and purification of thioesterase ScoT. 1

Type II thioesterase ScoT was obtained as an N-terminal His6-tagged proteins 2

expressed in E. coli. The recombinant protein was found mostly in the insoluble fraction of 3

cell lysate in all induction conditions tested (IPTG concentration 0.1 mM and 0.01 mM, 4

induction at OD600 = 0.6 and 1.0, incubation temperatures 37°C, 28°C and 9°C). Low 5

incubation temperature and low IPTG concentration slightly increased the amount of ScoT 6

protein in the soluble fraction of cell lysate. Finally, induction with 0.01 mM IPTG at 7

OD600 = 1.0 and overnight incubation at 9°C was chosen for large scale protein purification. 8

Since enzymatic measurements were undertaken in this study, isolation in native conditions 9

was chosen in spite of low yield, to avoid denaturation step required for protein isolation from 10

inclusion bodies. ScoT protein was purified from crude cell extract by nickel affinity 11

chromatography followed by anion exchange chromatography on Q Sepharose. Figures of 12

SDS PAGE gels are shown in supplemental material. Approx. 100 µg of purified protein was 13

obtained from 1 liter of culture. Problems with insolubility and low yields of recombinant 14

TE IIs were reported also by Schwarzer et al. (36). 15

16

Substrate specificity of thioesterase ScoT 17

Aberrant decarboxylation of polyketide extender units such as malonate, 18

methylmalonate and ethylmalonate gives rise to acetyl, propionyl and butyryl residues 19

attached to ACP domains of PKS proteins and blocking the activity of the entire 20

multienzyme. Hydrolytic activity of the TE II ScoT was investigated using substrates 21

representing these acyl chains. Two groups of substrates were used: p-nitrophenyl (p-NP) 22

esters and N-acetylcysteamine (NAC) thioesters. Reaction of hydrolysis proceeds via two 23

steps: acylation and deacylation. NAC thioesters are good structural analogues of natural 24

thioesterase substrates – acyl groups attached to 4’-phosphopantetheine of ACP. They reflect 25

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well the acylation step of the hydrolysis reaction. More reactive p-NP esters are useful to 1

study the deacylation step and have been widely used in studies of other thioesterases (16, 44, 2

46). Progress of hydrolysis was monitored by spectroscopy. Determined kinetic constants are 3

given in table 2. 4

Plots of the initial reaction velocity of p-NP esters hydrolysis versus substrate 5

concentration are given in Fig. 1A. Due to limited solubility of p-NP esters in water it was 6

impossible to achieve substrate-saturating conditions and to determine the kcat and Km values. 7

The specificity constant kcat/Km was used to compare the activity of ScoT towards different 8

substrates. The kcat/Km values for each substrate were calculated from the linear slope of each 9

plot at low substrate concentration. Of the three p-NP esters, propionate was the best substrate 10

for the thioesterase. It was hydrolyzed 4.5 and 7 times more efficiently than acetate and 11

butyrate, respectively, as shown by comparing the specificity constants (table 2). 12

Plots of the initial reaction rates of hydrolysis of NAC thioesters versus substrate 13

concentration are given in Fig. 1B. Kinetic constants kcat and Km were calculated by fitting 14

experimental data to the Michaelis-Menten equation by non-linear regression using the 15

SigmaPlot software (table 2). Results obtained for NAC thioesters were consistent with those 16

for p-NP esters. Higher specificity constants observed for p-NP esters relative to NAC 17

thioesters (approx. 20-fold) can be attributed to the high reactivity of p-nitrophenol as a 18

leaving group. NAC propionate was hydrolysed much more readily than the other substrates. 19

The specificity constant for propionate was 7 and 13 times higher than for acetate and 20

butyrate, respectively. There are no big differences between Km values for acetate and 21

butyrate which suggests that initial binding of these substrates involves similar interactions. 22

Lower Km value for propionate indicates higher affinity of the enzyme to this compound. 23

Significant differences are observed between kcat constants, which reflect differences in the 24

rate of the chemical steps subsequent to substrate binding. 25

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1

Active site investigation 2

By sequence comparison and predicted secondary structure homology search ScoT 3

protein was assigned to the family of thioesterases (PF00975) from the clan of α/β hydrolases 4

(CL0028) (Pfam database). The proteins belonging to α/β hydrolases (lipases, esterases, 5

dehalogenases, epoxide hydrolases and others) have conserved structural features despite low 6

or even no sequence homology (17, 19, 31). The canonical α/β hydrolase fold is described as 7

an eight-stranded, mostly parallel β sheet surrounded on both sides by α helices (Fig. 2A). 8

Active sites of α/β hydrolases consist of the catalytic triad Nu-Ac-His (where Nu = 9

nucleophile, usually Ser; Ac = acid, usually Asp). The nucleophile is located in a tight turn 10

between strand β5 and helix αC called the “nucleophilic elbow” within a consensus sequence 11

Sm-X-Nu-X-Sm (where Sm = small residue, usually Gly; X = any residue). Histidine is 12

located on a loop after strand β8. The location of the acidic residue is the most variable: in the 13

canonical structure it is after strand β7. 14

Amino acid residues which may form the catalytic triad of ScoT thioesterase were 15

proposed on the basis of conservative sequence motifs identification (Fig. 3) and analysis of 16

two structural models. Model 1 was created by the Phyre software (protein homology/analogy 17

recognition engine, an improved version of 3D-PSSM, www.sbg.bio.ic.ac.uk/phyre/) which 18

performs predicted secondary structures matching and is able to find distant structural 19

homologues even with no sequence similarity (22). In case of model 2, proteins with the 20

highest sequence homology were selected by searching data bases on PDB and FUGUE 21

servers and their structures were used to build the model template. Secondary structure 22

topology diagrams representing models 1 and 2 are shown in fig. 2B and C, respectively. 23

Both models include the minimal set of α helices and β strands of the α/β hydrolase 24

fold (β3 – β7, αA – αD). The nucleophilic residue of ScoT is probably Ser90 found within the 25

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conservative sequence G-X-S-X-G typical for thioesterases and acyltransferases – in both 1

models this residue is located on the turn between strand β5 and helix αC. The two models 2

indicate different histidine residues located on a loop after strand β8 which could be involved 3

in the catalytic triad. These are His224 (model 1) and His210 (model 2). After the predicted 4

strand β7 there are two acidic residues: Asp194 and Asp196. One of them could potentially be 5

the acidic component of the catalytic triad. These five amino acids were selected for site-6

directed mutagenesis (Fig. 3). 7

Eight mutants carrying single substitutions (S90A, S90C, H210A, H210Y, H224A, 8

H224Y, D194N, D196N) and one double mutant (DD194/196NN) were constructed by 9

overlap extension. The double mutant was designed to exclude potential functional 10

replacement of one Asp residue by the other. ScoT_S90A, ScoT_S90C and ScoT_H210Y 11

were isolated using the same procedure as for wild type ScoT protein. ScoT_ H224Y was 12

expressed as a fusion protein with glutathione-S-transferase (GST). The remaining mutant 13

proteins were found in the insoluble fraction of E. coli lysate. Probably the mutations had a 14

negative effect on the process of protein folding. 15

Since propionate derivatives were the best substrates for the wild type ScoT, p-NP 16

propionate and NAC propionate were chosen for the enzymatic activity testing of the mutants. 17

Plots of the initial reaction rates versus substrate concentration are shown in Fig. 4. Calculated 18

catalytic constants are given in table 3. Replacement of serine 90 by alanine completely 19

abolished the hydrolytic activity of ScoT. The mutant with cysteine instead of serine retained 20

residual activity (approx. 10% of kcat/Km value). This leads to the conclusion that indeed 21

Ser90 is the nucleophilic residue of the catalytic triad. Obtained results indicate that the 22

second element of the catalytic triad is histidine 224, and not histidine 210. The activity of the 23

protein with H210Y mutation was close to that of wild type ScoT protein, and the activity of 24

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ScoT_H224Y mutant was much lower (40% of kcat/Km value for p-NP propionate and 55% 1

for NAC propionate). 2

3

Acylation of thioesterase. 4

In order to capture the acyl-enzyme intermediate, the technique of ESI-MS was used. 5

The reaction mixtures of ScoT with p-NP acetate, propionate and butyrate were set up, diluted 6

with a solution of acetonitrile/water/formic acid and introduced into the ion source. 7

Initial analysis of the mass spectrum of ScoT protein without any substrates added 8

revealed a presence of two major compounds a and b differing by 178 Da (Fig. 5A). 9

Determined molecular weight of the main species (compound a) was 30 718.48 ± 0.44 Da 10

(molecule [M+H]+) which is in agreement with expected molecular mass of recombinant 11

ScoT protein lacking the N-terminal methionine (30 717.98 Da). The second peak (b) 12

represented the same protein with a gluconic acid residue attached to its N-terminus. 13

Spontaneous α-N-gluconoylation is a commonly found posttranslational modification of His-14

tagged proteins expressed in E. coli and causes mass increase by 178 Da (12). A small amount 15

of acetylated ScoT was also present in the protein sample (compound e, with mass greater by 16

42 relative to compound a). 17

When p-NP esters were added, formation of acyl-enzyme was visible, as exemplifed 18

by the reaction with p-NP butyrate (Fig. 5B). Two additional peaks can be noticed 19

representing mass increase by 70 of the two forms of ScoT (native and gluconoylated, 20

compounds c and d, respectively). 21

Mutated thioesterase with serine 90 replaced by alanine was also subject to ESI-MS 22

analysis. Similarly as in case of wild type protein, two forms of ScoT_S90A protein were 23

found (Fig. 5C): compound a’ (lacking the N-terminal methionine) and compound b’ 24

(demethionylated and gluconoylated). When ScoT_S90A protein was incubated with p-NP 25

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butyrate formation of acyl-enzyme was not observed (Fig. 5D). This experiment confirmed 1

that the hydrolysis reaction proceeds through the acylation of Ser90 which is the active site of 2

thioesterase ScoT. 3

4

DISCUSSION 5

6

Thioesterase ScoT hydrolyses acyl chains and shows preference for propionate. 7

As shown by our study, thioesterase ScoT from S. coelicolor A3(2) is able to 8

hydrolyse acetyl, propionyl and butyryl residues which can block ACP domains. The 9

recombinant protein hydrolyzed all tested substrates at significant rates. This is consistent 10

with its editing function suggested by a complementation study in S. fradiae (25). The 11

enzyme showed clear preference for propionate. Specificity constants for propionate 12

hydrolysis were several times higher than for acetate. Butyrate residue was hydrolised the 13

slowest (kcat/Km for butyrate approx. two times lower than for acetate). 14

Michaelis constants and katalytic constants could only be determined for NAC 15

thioesters. Km value for propionate is slightly lower than for acetate and butyrate, which 16

suggests that ScoT binds this substrate with higher affinity. However, greater differences in 17

the reaction rates are associated with further steps, as indicated by the differences between kcat 18

values (table 2). 19

Determined kinetic constants can be directly compared to the parameters of TylO 20

thioesterase from tylosin synthase of S. fradiae which was tested on the same substrates (16). 21

ScoT was found more active than TylO. In both groups of substrates (p-NP and NAC 22

derivatives) kcat/Km values of ScoT for acetate and propionate were an order of magnitude 23

greater than corresponding values of TylO (Fig. 6). By comparing the parameters determined 24

for NAC propionate hydrolysis by ScoT (kcat = 450 min-1

, Km = 34 mM) and TylO 25

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(kcat = 29.2 min-1

, Km = 37,9 mM) it can be concluded that the differences are associated 1

mostly with the deacylation step of hydrolysis (significant differences between kcat values). 2

As suggested by Kim et al. (2002) (23), high Km values (>100 µM) indicate that TE II 3

operates in vivo under non-saturating conditions and is mostly dissociated from the PKS, 4

which enables it to scan all modules in search of stalled residues. 5

The same acyl chains attached to the phosphopanthetheine of an ACP domain were 6

tested as substrates for TE II (PikAV) from picromycin biosynthetic cluster of S. venezuelae 7

(23). Thioesterase ScoT exhibited greater differences in specificity for substrates with 8

different chain lengths than the thioesterases TylO and PikAV (Fig. 6). The order of substrate 9

preferences was also different in the three cases. Polyketide synthase Cpk from S. coelicolor 10

A3(2) utilises only malonyl-CoA as extender units, as predicted by sequence analysis of AT 11

domains (32), therefore theoretically only acetyl residues can be generated as products of 12

aberrant decarboxylation. However, when keeping in mind, that mispriming of ACP domains 13

can also give rise to blocking residues, activity towards other acyl chains is not surprising. 14

Different substrate specificities of ScoT compared to TylO and PikAV is probably associated 15

with their different structures, especially different shape of the substrate binding pocket. The 16

active site of ScoT seems to have some specific features which make it fit particularly well 17

the two-carbon chain of propionate, however, detailed structural studies would be necessary 18

to understand them. 19

Acyl residues which are starter units for polyketide biosynthesis are also possible 20

substrates for TE IIs. The loading module of Cpk synthase includes KS domain with 21

glutamine in the active site which probably decarboxylates malonyl residue incorporated by 22

AT domain to give acetyl starter unit (1). Obtained results lead us to speculation that ScoT 23

may have a role in control of starter unit by preventing accidental propionate incorporation. 24

To prove this hypothesis it is necessary to solve the chemical structure of the polyketide 25

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produced by the Cpk synthase and to identify the potential shunt product arising from 1

propionate starter. 2

High activity of ScoT for propionate can explain results of our earlier experiments. 3

When scoT gene from S. coelicolor A3(2) was used to complement tylO disruption mutant of 4

S. fradiae, polyketide production was restored up to 50% of the level of wild type strain (25), 5

while complementation with the native thioesterase gene tylO restored polyketide production 6

to nearly 100% (6). Starter unit for tylosin is propionate. One of the reasons that the strain 7

complemented with scoT gene obtained lower level may be excessive removal of propionate 8

starter unit from tylosin synthase. Genes used for complementation were expressed from a 9

strong, constitutive promoter ermE*p. Perhaps decreasing expression of ScoT thioesterase 10

would lead to higher polyketide production. 11

Chromosomes of Streptomyces and some other bacteria often contain several PKS and 12

NRPS gene clusters. The foreseen mechanisms preventing unwanted results of interactions 13

between synthases and TE IIs from different clusters may include “fine tuning” of kinetic 14

parameters of the enzymes, substrate specificity, specificity for different ACP (PCP) domains, 15

temporal and spatial regulation of expression. 16

The balance reached by evolution is disturbed by “man-made” modifications of 17

megasynthases (PKS, NRPS), construction of hybrid enzymes and their heterological 18

expression. Inclusion of a proper editing enzyme in the system is one of important aspects of 19

such experiments. There is also a possibility of interactions between the introduced synthase 20

and native TE IIs of the host organism. Brown et al. (4) observed that the main product of the 21

first subunit of DEBS expressed in S. coelicolor was the compound resulting from 22

incorporation of acetate instead of propionate. All three DEBS subunits expressed in 23

S. coelicolor produced 6-deoxyerythronolide B (6-dEB) with a significant addition of 15-nor-24

6dEB (arising from acetate starter), even when the TE II from DEBS was included (20). It 25

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seems probable, that high activity of ScoT in the host cells could contribute to this effect by 1

removal of propionate from the loading module of DEBS. 2

3

Active site of thioesterase ScoT 4

On the basis of theoretical sequence analysis we suggest, that ScoT protein belongs to 5

α/β hydrolases. Recently a three-dimensional structure of TE II involved in the biosynthesis 6

of nonribosomal peptide surfactin of Bacillus subtilis was resolved based on NMR titration 7

experiments (24). Surfactin TE II was found to have a typical α/β hydrolase fold. Several 8

crystal structures resolved so far showed that TE domains (type I thioesterases) of PKS (43) 9

and NRPS (5) also have the α/β hydrolase fold. However, important structural differences can 10

be noticed when comparing type I and II TEs, which explain their different roles. TE domains 11

form deep substrate channels which accomodate the whole polyketide/peptide products and 12

facilitate their cyclization while the open an shallow active site cavity of TE II can 13

accomodate only small acyl substrates. 14

Amino acids which may constitute the catalytic triad of the thioesterase ScoT (Ser-15

His-Asp) were proposed and exchanged by site directed mutagenesis. Our results show that 16

Ser90 is the active site of the enzyme. Substitution of this amino acid with alanine caused 17

complete inactivation of the enzyme. The same effect was observed when serine was replaced 18

with alanine in type II thioesterases (from rat FAS (41), surfactin NRPS (27) and 19

nanchangmycin PKS (29)), TE domains (of pikromycin PKS (30) and chicken FAS (33)) and 20

many other α/β hydrolases. 21

Substitution of Ser90 with cysteine gave a protein with low activity in comparison to 22

wild type enzyme (not more than 10% of kcat/Km value). Different results of such a mutation 23

can be found in the literature. For example, S101C mutation in TE domain of FAS caused 24

only 50% decrease of activity (33). Similarly, rat TE II with S101C mutation and TE II from 25

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surfactin synthetase with S86C mutation retained relatively high activities (27, 41). On the 1

other hand, activity of many α/β hydrolases dropped by several orders of magnitude on 2

replacement of serine with cysteine (18, 21, 26). The sulfhydryl group is a stronger 3

nucleophile than the hydroxyl group, therefore often the observed decrease of activity 4

associated with Ser => Cys substitution is associated with deacylation becoming the rate 5

limiting step (26). The atomic radius of sulphur is larger than that of oxygen. This probably 6

leads to changes in the geometry of the active site and in some cases disables the charge relay 7

system. 8

The role of Ser90 as the active site was further confirmed by the ESI-MS experiment. 9

Acyl-enzyme formation was observed when p-NP substrates were added to wild type ScoT 10

protein and not when the mutated protein ScoT_S90A was used. 11

Two different histidines (210 and 224) were indicated by two structural models of 12

ScoT protein as possible elements of the catalytic triad and each of them was mutated. 13

Initially they were replaced with alanine, but it was impossible to isolate the mutated proteins 14

from the soluble fraction of E. coli cell lysate. In the second round of the experiment, 15

histidine residues were replaced with tyrosine. Unexpectedly, enzymatic activities of both 16

H210Y and H224Y mutants were fairly high (kcat values determined for p-NP propionate 17

were, respectively, 77% and 33% of kcat of wild type ScoT). Since the effect of His => Tyr 18

substitution in the position 224 is more pronounced than in the position 210, His224 is 19

indicated as part of the catalytic triad. So far, reported mutations of histidines from catalytic 20

triads caused inactivation or large decrease of activity even if they were replaced with another 21

basic amino acid or tyrosine (2, 9, 27, 29, 45). A typical pKa of Tyr side chain is 10.0 (40). 22

Theoretically, this residue should remain uncharged at pH 7.5. Perhaps local conditions in the 23

active site of ScoT lead to lowering the pKa and allow ionisation of tyrosine. The resultant 24

negative charge might function as proton acceptor and in this way replace histidine. 25

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The third element of the catalytic triad remains unknown, since we failed to isolate 1

mutants of Asp194 and Asp196 in native conditions from the soluble fraction of E. coli cell 2

lysate. 3

Thioesterase ScoT from Streptomyces coelicolor A3(2) characterised here is unique 4

among the other TE IIs (from macrolide producing PKSs) tested so far due to its 5

exceptionally high activity for propionate. This feature could be exploited if propionate 6

residues posed a problem, and should also be kept in mind when using S. coelicolor as a host 7

for hybrid PKS expression. We propose, that thioesterase ScoT has an editing function similar 8

to that of TylO and also prevents inclusion of propionate starter unit by Cpk polyketide 9

synthase. We also conclude that this enzyme is an α/β hydrolase with Ser90 and His224 in the 10

active site. 11

12

ACKNOWLEDGEMENTS 13

14

We thank P. Jakimowicz for help in model construction and analysis and for advice on 15

protein isolation. We are grateful to M. Jon for performing mass spectroscopy measurements. 16

This work was supported by the Ministry of Scientific Research and Information (grant 17

3P04B 004 29). 18

19

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FIGURE LEGENDS 1

2

Fig. 1. Rate versus substrate concentration plots for the hydrolysis of p-nitrophenyl esters (A) 3

and N-acetylcysteamine thioesters (B) by thioesterase ScoT. Data points represent mean ± 4

SD. 5

6

Fig. 2. Topology diagrams of α/β hydrolases. Cylinders – α helices, arrows – β strands. 7

Shaded symbols represent the minimal set of α and β structures. (A) Canonical α/β hydrolase 8

fold. Catalytic triad amino acids are marked with dots. (B) Predicted topology of secondary 9

structures of ScoT tioesterase representing model 1. (C) Predicted topology of secondary 10

structures of ScoT tioesterase representing model 2. In panels B and C amino acids selected 11

for site directed mutagenesis are marked with dots. 12

13

Fig. 3. Alignment of ScoT aminoacid sequence with type II thioesterases associated with 14

bacterial polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and rat fatty 15

acid synthase (FAS). Amino acids selected for mutagenesis are marked with asterisks and 16

respective substitutions are listed above. RifR – TE II from rifamycin synthase Amycolatopsis 17

mediterranei (AAG52991), TEII_roch – predicted TE II S. rochei (NP_851508), TEII_aver - 18

predicted TE II S. avermitilis (NP_821582), Plm8 - TE II from phoslactomycin B synthase 19

Streptomyces sp. HK803 (AAQ84143), MonAX – TE II from monensin synthase 20

S. cinnamonensis (AAO65810), ORF5_tyl – TE II from tylosin synthase S. fradiae 21

(AAA21345), PikAV_ TE II from pikromycin synthase S. venezuelae (AAC69333), 22

ORF3_ery – TE II from DEBS Saccharopolyspora erythraea (CAA42928), GrsT – TE II 23

from gramicidin S synthetase Brevibacillus brevis (AAA58717), SrfA4 – TE II from surfactin 24

synthetase Bacillus subtilis (CAA49819), rat – TE II from rat fatty acid synthase (P08635). 25

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1

Fig. 4. Rate versus substrate concentration plots for the hydrolysis of p-NP propionate (A) and 2

NAC propionate (B) by ScoT thioesterase mutants. Data points represent mean ± SD. 3

4

Fig. 5. Detection of acyl-enzyme intermediate of the hydrolysis reaction catalysed by 5

thioesterase ScoT. (A) ScoT protein. (B) ScoT protein incubated with p-NP butyrate. (C) 6

ScoT_S90A protein. (D) ScoT_S90A protein incubated with p-NP butyrate. Both ScoT and 7

ScoT_S90A lack the N-terminal methionine. Peaks marked with letters represent the 8

following compounds: a – ScoT, b – gluconoylated ScoT, c – butyrylated ScoT, d – 9

butyrylated and gluconoylated ScoT, e – acetylated ScoT, a’ – ScoT_S90A, b’ – 10

gluconoylated ScoT_S90A 11

12

Fig. 6. Comparison of specificity constants (kcat/Km) of type II thioesterases ScoT 13

(S. coelicolor), TylO (S. fradiae) and PikAV (S. venezuelae) for different acyl residues 14

attached to N-acetylcysteamine (acyl-NAC) or acyl carrier protein (acyl-ACP). kcat/Km (M-1

s-

15

1) values are given under the graphs . Data for TylO and PikAV were taken from references 16

16 and 23, respectively. 17

18

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TABLES 1

2

Table 1. List of oligonucleotides used for site directed mutagenesis. Nucleotides different 3

from the wild type sequence of scoT are underlined and written in bold. 4

5

primer sequence (5’=>3’)

S90A-F C TTC GGG CAC GCC ATG GGC GCG

S90A-R CGC GCC CAT GGC GTG CCC GAA G

S90C-F C TTC GGG CAC TGC ATG GGC GCG

S90C-R CGC GCC CAT GCA GTG CCC GAA G

H210A-F G TGG CGC GAG GCC ACC ACC GCC

H210A-R GGC GGT GGT GGC CTC GCG CCA C

H210Y-F G TGG CGC GAG TAT ACC ACC GCC

H210Y-R GGC GGT GGT ATA CTC GCG CCA C

H224A-F G CCG GGC GGT GCC TTC TAT CTC

H224A- R GAG ATA GAA GCG ACC GCC CGG C

H224Y-F G CCG GGC GGT TAT TTC TAT CTC

H224Y-R GAG ATA GAA ATA ACC GCC CGG C

D194N-F C CTG GTC GGG AAT CAG GAC CCC

D194N-R GGG GTC CTG ATT CCC GAC CAG G

D196N-F GGG GAC CAG AAT CCC GTC GTT C

D196N-R G AAC GAC GGG ATT CTG GTC CCC

DD194/196NN-F C CTG GTC GGG AAT CAG AAT CCC

GTC GTT C

DD194/196NN-R G AAC GAC GGG ATT CTG ATT CCC

GAC CAG G

6

7

8

9

10

11

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Table 2. Kinetic constants of synthetic substrates hydrolysis by recombinant type II 1

thioesterase ScoT. 2

3

substrate kcat(min-1

) Km(mM) kcat/Km(M-1

s-1

)

acetate - - 788

propionate - - 3 567 p-NP

esters

butyrate - - 485

acetate 109± 7 56± 6 33

propionate 450± 67 34± 10 221 NAC

thioesters

butyrate 54± 8 52± 14 17

4

5

Table 3. Summary of ScoT mutant activities. 6

7

substrate ScoT kcat(min-1

) Km(mM) kcat/Km(M-1

s-1

)

wild type - - 3 567

S90A inactive inactive inactive

S90C 47± 8 2.09± 0.54 378

H210Y 540± 79 1.99± 0.48 4523

p-NP

propionate

H224Y 313± 41 3.65± 0.66 1428

wild type 450± 67 34± 10 221

S90A inactive inactive inactive

S90C 24± 5 24± 12 17

H210Y 320± 12 18± 2 303

NAC

propionate

H224Y 150± 6 20± 2 122

8

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S90A

S90C

DD194/196NN

D194N D196N

H210A

H210Y

H224A

H224Y

* * * * * ScoT (PKS) : 84 HAFFGHSMGALLAYEL (93 aa) GDQDPVVPVAAAAAWREHTTAGSDLRVLPGGHFYL 227 RifR (PKS) : 88 LALFGHSMGAIIGYEL (93 aa) GDHDPRVSVGEARAWEEHTTGPADLRVLPGGHFFL 231 TEII_roch (PKS) : 92 LALFGHSMGAVLAYEV (93 aa) GDSDPRLSIEEAAAWEDHTTGLTELQVFPGGHFYL 235 TEII_aver (PKS) : 91 IALFGHSMGASLAYEI (93 aa) GDDDPKVTVEEAAAWSGHTDGPFMLRVFPGGHFFL 234 Plm8 (PKS) : 91 MAFFGHSMGALLAFEV (93 aa) GADDPRTARDEAAAWQEHTTGDFALRVFPGGHFFI 234 MonAX (PKS) : 94 LAFFGHSMGAVLAYEV (93 aa) GDRDPQAPLDEVEAWQQHTEGPFDLKVFAGGHFYL 237 ORF5_tyl (PKS) : 87 VALFGHSMGAVVAYET (93 aa) GDADPNVTLPEAEAWRELTTGAFALRVFPGGHFYL 230 PikAV (PKS) : 93 LAFFGHSLGASVAFET (93 aa) GDRDPKAPLNEVAEWRRHTSGPFCLRAYSGGHFYL 236 orf3_ery (PKS) : 84 FALFGHSMGALIAYET (91 aa) GDADPLTATGDAERWLQHSVIPGRTRTFPGGHFYL 225 GrsT (NRPS) : 89 FAFLGHSMGALISFEL (94 aa) GKNDNGVTYQSLEAWREQTKREFSVCMYPGDHFFL 233 SrfA4 (NRPS) : 80 FVLFGHSMGGMITFRL (91 aa) GLDDKKCIR-DAEGWKKWAK-DITFHQFDGGHMFL 219 rat (FAS) : 95 FAFFGHSFGSYIALIT (98 aa) GSEDTIK---DIEGWQDLTSGKFDVHMLPGDHFYL 240

X 100% conservation X > 80% conservation X > 60% conservation

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1 Type II thioesterase ScoT from 2 3 propionate....

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