Enzymatic and Regulatory Properties of the Trehalose-6-Phosphate Synthase from the Thermoacidophilic Archaeon Thermoplasma Acidophilum

Biochimie 101 (2014) 215e220

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Research paper Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum

Yanyan Gao 1, Ying Jiang 1, Qiulei Liu, Ruiming Wang, Xinli Liu, Bo Liu*

College of Food and Bioengineering, Qilu University of Technology, Jinan, Shandong 250353, PR China article info abstract

Article history: Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the Received 12 November 2013 transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Accepted 27 January 2014 Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic Available online 4 February 2014 archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl ac- Keywords: ceptors. The optimal temperature and pH were found to be 60 C and pH 6, and the enzyme exhibited Trehalose-6-phosphate synthase notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and Enzyme regulation polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives pH and thermal stability b N-Loop sequence ethanol, EDTA, urea, DTT, SDS, -mercaptoethanol, methanol, isopropanol and n-butanol. Molecular Molecular modeling modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic ef- Thermoplasma acidophilum ﬁciency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases. Ó 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction 6-P) with the production of trehalose 6-phosphate (T6P) by the trehalose-6-phosphate synthase (TPS: EC 2.4.1.15). Subsequently, Trehalose is a non-reducing disaccharide in which the two T6P is dephosphorylated to produce trehalose by the trehalose 6- glycosyl moieties are linked together by an a,a-1,1 bond [1]. It exists phosphate phosphatase (TPP: EC 3.1.3.12) [3]. Furthermore, the in many organisms and can be used as a carbon and energy source TPS and TPP genes are fused together to encode a fused trehalose- in metabolism. Moreover, the sugar has remarkable stress protec- 6-phosphate synthase/phosphatase (TPSP) protein in almost all tion properties to serve as a protectant against various stress con- eukaryotic genomes, whereas the two genes are usually clustered ditions such as desiccation, temperature, salinity, alkalinity and in one operon but encoded separately in bacterial genomes. Inter- oxidation [2]. There are at least ﬁve pathways for trehalose estingly, the only characterized TPS/TPSP gene in archaeal genomes biosynthesis in Bacteria, whereas only the TPS/TPP pathway is from the hyperthermophilic crenarchaeon Thermoproteus tenax present in Eukaryotes. To date, four pathways (TPS/TPP, TreYeTreZ, shows a unique operon organization with a glycosyltransferase TreT and TreS) for trehalose biosynthesis have been found in (GT) and a mechanosensitive channel (MSC) gene, and also encodes Archaea. The TPS/TPP pathway, which is distributed in all three a TPSP fusion enzyme. The GT is required for the bifunctional ac- domains of life, consists of a two-step catalysis mechanism. Firstly, tivity of the TPSP. The authors also suggested a monophyletic origin the glycosyl is transferred from UDPG to glucose 6-phosphate (Glc- of eukaryotic and prokaryotic fused TPSPs during evolution [4]. Although many TPSs/TPSPs have been well characterized from Eukaryotes and Bacteria such as fungi, plants and bacteria [5e7],

Abbreviations: (UDPG), UDP-glucose; (Glc-6-P), glucose 6-phosphate; (TPS), current knowledge about TPS in Archaea is comparatively lacking. trehalose-6-phosphate synthase; (TPP), trehalose-6-phosphate phosphatase; Rao et al. [8] reported the crystal structure of a TPP from the (TPSP), trehalose-6-phosphate synthase/phosphatase; (T6P), trehalose 6- thermoacidophilic euryarchaeon Thermoplasma acidophilum, and phosphate; (ADPG), ADP-glucose; (GDPG), GDP-glucose; (GT), glycosyltransferase; its kinetics properties suggested that the enzyme is involved in the (MSC), mechanosensitive channel. TPS/TPP trehalose biosynthesis pathway. In the genome of T. acid- * Corresponding author. Tel.: þ86 13806402782; fax: þ86 531 89631192. E-mail address: [email protected] (B. Liu). ophilum, an ORF (TA1210) was found to be adjacent to the TPP gene 1 These authors contribute equally to this paper. (TA1209), which was assumed to encode the TPS. Our research aims

Fig. 1. Alignment of the amino acid sequences of taTPS with some of its homologous proteins. The alignment was carried out using the Clustal W program. Conserved residues are indicated by an asterisk below the alignment, and single and double dots represent amino acids with semi-conservative and conservative characteristics. Gaps introduced during the alignment process are indicated as dashes. The conserved N-loop sequence of taTPS is underlined, and the invariable glycosyl acceptor and donor interactive residues (Arg9 and Gly23) in the N-loop region are boxed. The conserved residues (Arg9, Trp45, Tyr81, Trp90, Asp135 and Arg284) involved in glycosyl acceptor binding are indicated by open arrows, and residues (Gly29, His159, Arg246, Lys251, Asp345 and Glu353) involved in glycosyl donor binding are indicated by ﬁlled dots. For comparison, the TPP domain of the TPSP from Y. Gao et al. / Biochimie 101 (2014) 215e220 217 were to investigate the enzymatic and regulatory properties of this enzyme producing 1 mmol T6P per minute. All enzyme assays were protein. It is the ﬁrst report about the biochemical and structural performed in triplicates and the data were averaged. properties of TPS from euryarchaeon.

2.4. Effects of temperature and pH on enzyme activity and stability 2. Materials and methods The effect of temperature on taTPS activity was determined at 2.1. Chemicals, enzymes, bacterial strains and plasmid temperatures ranging from 30 to 80 C according to the standard a enzyme assay method. The thermostability was determined by The Escherichia coli strains DH5 and BL21-CodonPlus (DE3)-RIL preheating the enzyme in 20 mM sodium phosphate (pH 6) at 60, and plasmid pET15b were from Novagen. All molecular manipula- 70 and 80 C for different lengths of time, respectively, and the tion enzymes and kits were from Takara Bio. UDPG, ADPG, residual activities were measured. GDPG, Glc-6-P, phosphoenolpyruvate, pyruvate kinase, lactic The optimal pH was determined in various pHs of 20 mM buffer acid dehydrogenase, glucosamine-6-phosphate, fructose-6-phos as follows: Na2HPO4/citric acid (pH 4e5), sodium phosphate (pH phate, mannose-6-phosphate, heparin sodium and chondroitin 6e7) and Tris/HCl (pH 8e10). The pH stability was determined by e sulfate were obtained from Sigma Aldrich. Unless stated, all other preheating the enzyme in various pH buffers at room temperature chemicals were analytical grade and obtained from Sangon Biotech for different lengths of time, respectively, and the residual activities (Shanghai, China). were measured.

2.2. Cloning, expression and purification of the T. acidophilum TPS 2.5. Substrate specificity, effects of metal ions, polyanions and To express the encoded protein of the gene TA1210 (GenBank: various additives CAC12335), the nucleotide sequence of this gene was synthesized by fi the Taihe Biotechnology Co., Ltd (Beijing, China), and its codon To study the substrate speci city, ADPG and GDPG were was optimized for E. coli expression. Two primers [sense (50-TCA- substituted for UDPG and alternative glycosyl acceptors glucos- CATATGAAATACATCGTTGTTTCTTCCCG-30) and antisense (50- amine-6-phosphate, fructose-6-phosphate, mannose-6-phosphate TGGTCGACTCAAGTAACAGTGCGATCGTTG-30), underlined nucleo- were in replace of glc-6-P, respectively. To investigate the effects tides indicated the NdeI and SalI restriction enzyme sites respec- of metal ions on the enzyme activity, the reaction was performed tively] were used to amplify the gene by PCR. The PCR product was under the standard enzyme assay conditions except that 20 mM Tris/HCl (pH 7) was used instead of sodium phosphate, and 10 mM digested by NdeI and SalI and then cloned into plasmid pET15b. The þ þ þ recombinant plasmid was designated as pET15b-taTPS and verified monovalent metal ions Na ,K and Li and divalent metal ions 2þ 2þ 2þ 2þ 2þ 2þ by DNA sequencing. The N-loop truncated mutation of taTPS was Zn ,Ba ,Ni ,Co and Mn were added instead of Mg , e generated with the clone method described above except that the respectively. The effects of polyanions were measured by adding 0 0 m e m sense primer was replaced by 5 -GTACATATGGTTGCGACCGCACT 2 g heparin sodium or 0 0.8 g chondroitin sulfate in the stan- GCGCCGTG-30. For overexpression of the recombinant protein, the dard reaction mixtures. Various additives ethanol, EDTA, urea, DTT, b recombinant plasmid was transformed into E. coli BL21-CodonPlus SDS, -mercaptoethanol, methanol, isopropanol, n-butanol and (DE3)-RIL. An overnight culture in LB medium at 37 C was diluted guanidine hydrochloride were added respectively to study their possible effect on the enzyme activity. 1:100 and grown until the OD600 reached 0.5, and then induced with 0.5 mM IPTG at 30 C for 12 h. The purification procedure of recombinant protein was as described previously [9]. The purified protein was analyzed by SDS-PAGE and gel filtration with Superdex 2.6. Kinetic properties 200Ô 10/300 GL column (Amersham) equilibrated in 25 mM glycine/ NaoH, 5% glycerol (v/v), 500 mM NaCl buffer (pH 10.5) at 0.5 ml/min. Kinetic properties were studied using six different substrate concentrations (0.5, 1, 1.5, 2, 2.5, 3 mM for UDPG and Glc-6P respectively) according to the standard enzyme assay except that 2.3. Standard enzyme assay of taTPS the initial reaction mixture was incubated at 60 C for 5 min. Kinetic parameters were determined from the rates by means of respective TPS activity was assayed by a colorimetric method as described LineweavereBurk plots. previously [10]. The 100 ml assay mixture contained 1 mM Glc-6-P, 1 mM UDPG, 10 mM MgCl2 and an appropriate amount of purified enzyme in 20 mM sodium phosphate (pH 6). After incubation at 2.7. Molecular modeling 60 C for 20 min, the mixture was boiled for 5 min and then centrifuged at 13,000 rpm/min for 10 min to remove the denatured The homology modeling of taTPS was generated with the Swiss proteins. The amount of released UDP in the supernatant was Model server (http://swissmodel.expasy.org/) using the TPS from measured in a reaction mixture containing 2.5 mM phosphoenol- E. coli (OtsA, PDB: 1GZ5, chain A) as the template, which is 33% pyruvate, 0.25 mM NADH, 50 mM Tris/HCl (pH 7.5), 3 U pyruvate sequence identity to taTPS. A multiple sequence alignment of taTPS kinase and 3 U lactic acid dehydrogenase. After incubation at 37 C with its homologous proteins was generated using the Clustal W for 30 min, the absorbance at 340 nm was determined as NADH program. The stereochemical quality of the model was evaluated by oxidation. The assay mixtures without Glc-6-P were used as blanks. the Verify 3D program with acceptable scores [11]. The modeled One unit of the enzyme activity was defined as the amount of structure was analyzed using the PyMOL software [12].

the hyperthermophilic crenarchaeon T. tenax is double lined. taTPS: TPS from T. acidophilum; T. tenax: TPSP from T. tenax; E. coli: TPS from E. coli; P. freudenreichii: TPS from Propionibacterium freudenreichii; S. cerevisiae: TPS1 (no fused TPS structure) from Saccharomyces cerevisiae trehalose synthase complex, D. melanogaster: DmTPS (fused TPSP structure) from Drosophila melanogaster, O sativa: OsTPS1 (fused TPSP structure) from Oryza sativa; A. thaliana: AtTPS1 (fused TPSP structure) from Arabidopsis thaliana. The accession numbers of the alignment proteins are stated in the result “Sequence analysis of taTPS” (2-column ﬁtting image). 218 Y. Gao et al. / Biochimie 101 (2014) 215e220

Thermoplasmatales archaeon A-plasma (EQB71681, 55%) and Pic- rophilus torridus (AAT43794, 55%). However, all these proteins have not been functionally characterized. Notably, the enzyme showed 40% identity with the only characterized TPSP (CCC81939) in Archaea from the hyperthermophilic crenarchaeon T. tenax [4]. Moreover, it also showed overall sequence identity to its eukaryotic and bacterial counterparts: the TPSs (OtsA) from E. coli (AAC74966, 33%) and Propionibacterium freudenreichii (CBL56737, 34%) [6,13], the TPS1 subunit of the four proteins of the Saccharomyces cerevisiae trehalose synthase complex (DAA07243, 31%) [14], the DmTPS from Drosophila melanogaster (ABH06633, 38%) [15], OsTPS1 from rice Oryza sativa (AEB53177, 32%) and AtTPS1 from Arabidopsis thaliana (CAA69879, 30%) [16,17]. It should be pointed out that all the proteins from the insect and plant exist as fusion enzymes (TPSP) which contain both a TPS and a TPP domain, although only the TPS activities are present in these enzymes. Sequence alignment showed many conserved amino acids which were involved in catalysis: the residues Arg9, Trp45, Tyr81, Trp90, Asp135 and Arg284 are involved in glycosyl acceptor binding, and Gly29, His159, Arg246, Lys251, Asp345 and Glu353 are involved in glycosyl donor binding. The conserved N-loop sequence was proposed to provide cross-talk between the glycosyl donor and acceptor domains (Fig. 1) [18,19].

3.2. Primary structure of taTPS

Fig. 2. Substrate specificity of taTPS. a Glycosyl donor specificity of taTPS. The maximal The recombinant protein was over-expressed in E. coli at high activity was defined as 100% level (7.33 U/mg). b Glycosyl acceptor specificity of taTPS. Glc-6-P: glucose-6-phosphate; Ga-6-P: glucosamine-6-phosphate; Fru-6-P: fructose- level and purified by metal chelating affinity chromatography 2þ 6-phosphate; Man-6-P: mannose-6-phosphate. The maximal activity was defined as (Ni -NTA column). The enzyme showed a molecular mass of about 100% level (7.41 U/mg) (1-column fitting image). 50 kDa in SDS-PAGE (Supplemental Fig. 1) and a peak corresponding to a molecular mass of about 100 kDa in gel filtration (data not shown), suggesting that the oligomeric state of the 3. Results enzyme was dimeric. It is reported that the SlTPS1 from Selaginella lepidophylla (which consists of an N-terminal TPS domain and a C- 3.1. Sequence analysis of taTPS terminal TPP domain) can aggregate as a dimer, tetramer or hex- amer [20], and the TPS from Mycobacterium tuberculosis exists as a In the genome of T. acidophilum, a gene TA1210 is adjacent to the tetramer [21]. characterized TPP gene and is suggested to encode the TPS involved in the trehalose biosynthesis pathway of this organism [8]. It en- 3.3. Substrate specificity codes a protein of 441 amino acids with a calculated molecular mass of 51.4 kDa (http://www.expasy.org/tools/pi_tool.html). The The enzyme could utilize various nucleoside diphosphate protein showed high sequence similarity to its archaeal homo- monosaccharides UDPG, ADPG and GDPG as catalytic donors, and logues from Thermoplasma volcanium (GenBank: BAB60419, 71%), maximal activity was found towards UDPG (7.33 U/mg). On the

Fig. 3. Effects of temperature and pH on taTPS activity. a The optimal temperature of taTPS. The maximal activity was deﬁned as 100% level (7.1 U/mg). b The optimal pH of taTPS (100%, 7.29 U/mg). c Thermostability of taTPS (100%, 6.57 U/mg). d pH Stability of taTPS (100%, 6.06 U/mg) (1-column ﬁtting image). Y. Gao et al. / Biochimie 101 (2014) 215e220 219 other hand, various phosphorylated monosaccharides Glc-6-P, Table 2 a glucosamine-6-phosphate, fructose-6-phosphate and mannose-6- Effects of various additives on taTPS activity. phosphate could be used as catalytic acceptors, with maximal ac- Chemicals Relative activity (%) tivity towards Glc-6-P (7.41 U/mg) (Fig. 2a and b). No additive 100 Methanol 70 Ethanol 72 3.4. Optima of temperature and pH, temperature and pH stability SDS 73 Isopropanol 86 The enzyme was optimally active at 60 C and pH 6, respectively EDTA 85 (Fig. 3a and b). It displayed more than half of the maximal activity at DTT 73 e e Urea 85 50 80 C and pH 5 7. Notably, the enzyme was thermostable at n-Butanol 52 high temperature 60, 70 and 80 C and more than 30% of the b-Mercaptoethanol 91 original activity remained after heat-treatment at 70 C for 10 h. Guanidine hydrochloride 37 The half-life of the enzyme activity at 60 C was 6 h (Fig. 3c). a taTPS activity was determined as described in Materials and method. The Furthermore, the enzyme was considerably stable in acid or basic activities were expressed as the percentage of the activity without additive conditions with more than half of the activity remaining after added (100%, 7.3 U/mg). treatment at pH 2 and 9 for 6 h, respectively (Fig. 3d). These results demonstrate the characteristic thermal and pH tolerance of TPS 3.7. Molecular modeling and roles of N-loop sequence from Archaea [22].

The structure of taTPS was modeled with the TPS (OstA) from 3.5. Regulation of the activity by polyanions and metal ions E. coli as the template (Supplemental Fig. 3). The modeled structure is composed of two b/a/b folds which consist of the glycosyl donor Many studies reported that polyanions could regulate TPS ac- (UDPG) and glycosyl acceptor (Glc-6-P) binding sites, respectively. tivity [23,24]. It turned out that 0e2 mg heparin could stimulate the The conserved N-loop region, being located at their interface, was activity of taTPS, although the activity decreased while the con- proposed to mediate communication between the two domains centration of heparin was above 0.6 mg(Supplemental Fig. 2a). and provide the location for catalysis [18,19]. The invariable resi- Moreover, 0e1 mg chondroitin sulfate could also stimulate the dues Arg9 and Gly29 in the N-loop region could interact with Glc-6- enzyme activity, and the maximal activation was found at the P in the N-terminal glycosyl acceptor domain and UDPG in the C- concentration of 0.4 mg(Supplemental Fig. 2b). The regulatory terminal glycosyl donor domain, respectively. properties of taTPS activity by polyanions were similar to those of The N-loop truncation mutation of taTPS was expressed and TPSs from S. cerevisiae, Mycobacterium smegmatis and Candida utilis puriﬁed as described. The deletion of N-terminal loop led to the [23e25]. Furthermore, the enzyme did not require divalent metal higher Km and lower Vmax values (0.34 mM and 8.45 U/mg for Glc- þ þ þ þ ions for catalysis, but Mg2 ,Zn2 ,Co2 and Mn2 could stimulate 6-P and 0.74 mM and 4.81 U/mg for UDPG) compared with those of enzyme activity and maximal activation was found in the presence wild type taTPS (0.27 mM and 12.46 U/mg for Glc-6-P and 0.21 mM þ þ þ þ of Mg2 . The addition of monovalent metal ions Na ,K and Li and 8.77 U/mg for UDPG), indicating the important roles of this had no effect on the enzyme activity (Table 1). domain in catalysis.

3.6. Effects of various additives 4. Discussion

> taTPS appeared to be resistant to the additives as follows ( 50% In this study, we cloned, expressed and characterized the TPS of the original activity remaining): organic solvents methanol, from the thermoacidophilic archaeon T. acidophilum. The enzyme ethanol, isopropanol and n-butanol (10%, v/v), reducing agents DTT showed broad substrate speciﬁcity, notable thermostability and b (10 mM) and -mercaptoethanol (10%, v/v), chelating reagent EDTA acid/basic stability, and remarkable resistance to organic solvents (10 mM), and denaturing agents urea (10 mM) and SDS (10%, w/v). and denaturants, which have not been described for TPS until now. However, the enzyme was partly inhibited by guanidine hydro- Considering the biotechnological advantages of extremozymes chloride (10 mM, 37% of the original activity remaining) (Table 2). such as improvement of the rate of reaction, avoiding contamina- These results indicate the inheritance of resistance of the archaeal tion and resistance to extreme conditions, the archaeal TPS could be TPS [26]. exploited to be useful in biotechnological applications, such as enzymatic synthesis of trehalose-6-phosphate or transgenic

Table 1 improvement of abiotic stress tolerance in organisms [27]. Regulation of the activity of taTPS by metal ions.a The relationship between the structure and function of TPS has been an interesting subject for decades. For enzymes involved in Metal ions Relative activity (%) the TPS/TPP pathway, only the crystal structures of a TPS (OstA) No ions 100 þ from E. coli [6], a TPP from T. acidophilum and a trehalose repressor Mg2 233 þ (TreR) from E. coli have been solved until now [8,28], and no three- Zn2 226 þ Co2 191 dimensional information is available for a TPSP fusion protein. þ Mn2 116 There are two domains in T. acidophilum TPP, the core domain has þ Ni2 79 an a b þ / hydrolase fold and resembles those of OstA and TreR, Ba2 62 þ whereas the cap domain is different [8]. Considering the trehalose Na 92 þ K 90 synthase complex of S. cerevisiae which is composed of TPS1, TPS2, þ Li 105 TPS3 and the regulatory subunit TSL1 [14], and the TPSP-GT complex from the hyperthermophilic crenarchaeon T. tenax which is a taTPS activity was determined as described in Materials and method. The activities were expressed as the percentage essential for the TPS activity of TPSP [4], very elaborate steric ar- of the activity without metal ions added (100%, 1.68 U/mg). chitectures must exist in these complexes to account for the fine 220 Y. Gao et al. / Biochimie 101 (2014) 215e220 tuning mechanism of trehalose synthesis. On the other hand, it is [9] Y. Hong, L. Wu, B. Liu, C. Peng, D.H. Sheng, J.F. Ni, Y.L. Shen, Characterization of reported that TPS and TPP can interact with each other [29], and the a glucan phosphorylase from the thermophilic archaeon Sulfolobus tokodaii strain 7, J. Mol. Catal. 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