Structural Characteristics and Function of a New Kind of Thermostable

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Structural Characteristics and Function of a New Kind of Thermostable Trehalose Synthase from Thermobaculum terrenum Junqing Wang, Xudong Ren, Ruiming Wang, Jing Su, and Feng Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02732 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 20, 2017

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Journal of Agricultural and Food Chemistry

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Structural Characteristics and Function of a New Kind of

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Thermostable Trehalose Synthase from Thermobaculum terrenum

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Junqing Wang, a, # Xudong Ren, a, # Ruiming Wang, a Jing Su ,a, * Feng Wang b

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a

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University of Technology, Jinan 250353, P.R. China.

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b

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University, Jinan, Shandong 250100, China

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#These authors contributed equally to the paper.

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Corresponding author: Jing Su

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Faculty of Light Industry, Province Key Laboratory of Microbial Engineering, Qilu

State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong

(Email: [email protected]. Tel: 86-531-88631076)

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1

ACS Paragon Plus Environment


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ABSTRACT: Trehalose has important applications in the food industry and

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pharmaceutical manufacturing. The thermostable enzyme trehalose synthase from

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Thermobaculum terrenum (TtTS) catalyzes the reversible interconversion of maltose

15

and trehalose. Here, we investigated the structural characteristics of TtTS in complex

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with the inhibitor TriS. TtTS exhibits the typical three domain glycoside hydrolase

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family 13 structure. The catalytic cleft consists of Asp202-Glu244-Asp310 and

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various conserved substrate-binding residues. However, among trehalose synthases,

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TtTS demonstrates obvious thermal stability. TtTS has more polar (charged) amino

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acids distributed on its protein structure surface and more aromatic amino acids buried

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within than other mesophilic trehalose synthases. Furthermore, TtTS structural

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analysis revealed four potential metal ion-binding sites rather than the two in a

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homologous structure. These factors may render TtTS relatively more thermostable

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among mesophilic trehalose synthases. The detailed thermophilic enzyme structure

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provided herein may provide guidance for further protein engineering in the design of

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stabilized enzymes.

27 28

Keywords: Thermobaculum terrenum, trehalose synthase, crystal structure,

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thermostability

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INTRODUCTION

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Trehalose is a kind of nonreducing disaccharide that is formed by α,α-1,1-linkage

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of two glucose molecules. It was first isolated from Claviceps purpurea and

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subsequently has been widely found across various organisms.1 In the literature,

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trehalose plays important roles as the carbon source and nitrogen source for organisms.

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It represents an important protectant during environmental stress such as cold,

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dehydration, heat, and oxygen stress.2–5 In some microorganisms such as

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Mycobacterium it plays an important role as a bacterial cell wall component.6,7

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Furthermore, because trehalose exhibits good water-holding capability and stress

40

resistance, it has many applications in the food, pharmaceutical, and cosmetic

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industries as a stabilizer and additive.8–11 However, the caveat of widespread trehalose

42

application is the high cost of its production.

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In order to reduce the production cost, investigators have focused on searching for

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highly efficient synthetic processes.12,13 At least five trehalose synthetic pathways are

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known in microorganisms. These pathways are composed of different enzyme

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systems, of which three are relatively well studied. The first pathway is the TPS-TPP

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pathway, which includes trehalose-6-phosphate synthase and trehalose-6-phosphate

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phosphatase.14,15 The second pathway also involves two enzymes, maltooligosyl

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trehalose synthase (TreY) and maltooligosyl trehalose trehalohydrolase (TreZ), and is

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termed the two-enzyme method for industrial trehalose production.16,17 This method

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uses starch as substrate and the conversion rate is higher than 80%. It produces less

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byproducts, glucose or maltose and is a main method for trehalose production . The

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third pathway, which only refers to trehalose synthase (TreS), can catalyze the

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reversible interconversion of maltose and trehalose in the absence of any coenzyme.

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This enzyme process has many advantages, such as a simple reaction, high substrate

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specificity, and low cost, and therefore offers good application prospects for trehalose

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production.18,19 However, TreS has shortcomings that limit its application; byproduct

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glucose is usually produced during the reaction process, and the isolation of trehalose

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is difficult. Therefore, it is important to analyze TreS three-dimensional structure and

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further modify the protein for production.

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TreS (EC 5.4.99.16) belongs to glycoside hydrolase family 13 (GH13). To date,

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several treS genes have been cloned from different organisms. The first treS gene was

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cloned from Pimelobacter sp. R48;18 subsequently, other microorganisms have also

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yielded treS genes, including Mycobacterium smegmatis,20 Pseudomonas stutzeri

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CJ38,21 Picrophilus torridus,22 Arthrobacter aurescens,23 Enterobacter hormaechei,24

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and Meiothermus ruber.25 These various microbial TreSs can be divided into

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structurally similar mesophilic and thermophilic enzymes. To date, the reported

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thermophilic TreSs are from Pimelobacter sp. R48,18 Picrophilus torridus,22

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Meiothermus

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Environments compatible with mesophilic TreS function are also suitable for the

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growth of a variety of microbes, and thus the use of this type of enzyme preparation

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of trehalose is often susceptible to bacterial contamination. The optimum temperature

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for these thermophilic TreSs is generally 60~80 °C, so they have good application

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prospects. Although the mesophilic and thermophilic enzymes are active at different

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temperatures, they share a conserved catalytic core (β/α)8 barrel and the same

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enzymatic catalysis mechanism.28–30 The interconversion of maltose and trehalose

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employs a double-displacement mechanism with a covalent glycosyl-enzyme

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intermediate;31 thus, the differences in thermal stability are mainly caused by minute

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structural differences. To date, there has been no report of a thermophilic TreS

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three-dimensional structure; therefore, it is highly desirable to obtain a

ruber,25

Thermus

thermophilus,26

and

4


Thermus

aquaticus.27

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three-dimensional structure for a thermostable TreS in order to study its thermophilic

82

mechanisms.

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To address this need, the first aim of the present study was to clone and express a

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thermostable TreS from Thermobaculum terrenum (ATCC BAA-798) (TtTS) in

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Escherichia coli BL21 (DE3). The temperature of recombinant enzyme optimal

86

activity was 45 °C, and it retained 80% of its initial activity after heat treatment at

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70 °C for 30 min. We further aimed to determine the crystal structure of the TreS

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complexed with the inhibitor TriS (Tris (Hydroxymethyl) aminomethane) and

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elucidate the determinant that affects the TtTS thermostability. The high-resolution

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structure of TreS from T. terrenum is essential for facilitating further protein

91

engineering.

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MATERIALS AND METHODS

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Bacterial strains, chemicals, media, and culture conditions. Escherichia coli

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BL21 (DE3) was used as the expression host and was cultured at 37 °C in lysogeny

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broth (LB). Enzymes for DNA amplification and restriction were purchased from

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Takara Bio Inc. (Kyoto, Japan). Glucose, maltose, and trehalose were purchased from

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Sigma (St. Louis, MO). Protein assay reagents and dyes were purchased from

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TransGen (Beijing, China). Crystal screens kits were purchased from Hampton

100

Research (Aliso Viejo, CA) and Emerald Bio (Bedford, MA). Other chemicals and

101

reagents were of analytical grade and purchased from Sigma (St. Louis, MO).

102 103

Cloning, Oligonucleotide-directed Mutagenesis, Protein Expression, and

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Purification. The treS sequence was synthesized according to the treS gene

105

(GenBank accession no. ACZ41252.1) from Thermobaculum terrenum (ATCC

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BAA-798) by TsingKe Company (QingDao, China). We amplified the treS sequence

107

by Pfu PCR with the primers F: catgtcCATATGAATGATGATCCGACGTG and R:

108

tcactcgagAGGCAGCTGTTCCTGTGG. The treS gene was inserted into the NdeI and

109

XhoI sites of pET-21b (Novagen, Madison, WI) in frame with a C-terminal 6× His-tag

110

and the resulting plasmid was transformed into E. coli BL21 (DE3) cells for TreS

111

overexpression. Three TtTs mutant combinations (R283G/Y287R/R291G, H534Y, and

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R283G/Y287R/R291G/H534Y) were produced using a one-step cloning kit (Vazyme

113

Biotech, Nanjing, China) and were confirmed by DNA sequencing (TsingKe, Qingdao,

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China). The mutagenesis primers are shown in Table S1.

115

E. coli BL21 (DE3) harboring the treS overexpression plasmid was grown in Luria

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Broth medium containing 100 µg/mL ampicillin until the OD600 reached 1.0. TreS

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overexpression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG)

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(0.12 mM, final concentration) and incubating overnight at 20 °C. Cells were

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subsequently harvested by centrifugation and lysed by ultrasonication in lysis buffer

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(25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.4 mM phenyl methane sulfonyl fluoride).

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After centrifugation at 28,000 × g for 45 min at 4 °C, the supernatant was applied to a

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Ni-NTA column (GE Healthcare, Little Chalfont, UK). The 6× His-tagged TreS was

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eluted with elution buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 250 mM

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imidazole). The protein was further purified by anion exchange on a Source-Q

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column (Source 15Q HR 16/10, GE Healthcare) and finally by size-exclusion

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chromatography (Superdex 200 10/300 GL, GE Healthcare). Purified TtTS was

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analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

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followed by Coomassie blue staining.

129 130

Enzymatic Activity Assays. The activity of TtTS was quantified by measuring

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the trehalose yield produced by maltose. The enzyme reaction system was as follows:

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20 mM Na2HPO4-NaH2PO4, buffer (pH 8.0), 150 mM maltose, and 1 µM TtTS in a

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final volume of 100 µL. Enzyme activity was assessed at 0.5, 0.75, 1, 1.5, 2, 2.5, 3,

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and 4 h and hourly until 14 h.

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The effects of temperature on enzyme activity were determined by varying the

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mixture reaction temperature from 30 °C to 80 °C for 8 h. The temperature-stability of

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TtTS was determined by incubation at temperatures of 40–80 °C for 30 min,

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respectively. This reaction mixture was then heated at 100 °C for 10 min to stop the

139

reaction. The effects of pH on TtTS activity were determined by performing the

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reactions in 20 mM acetic acid sodium acetate, Na2HPO4-NaH2PO4, or NH3-NH4Cl

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buffer systems, with pH ranges of 4.0–5.5, 6.0–8.5, and 9.0–11.0, respectively. TtTS

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activity was also assayed in the presence of metal ions and other chemical reagents (at

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1 mM and 10 mM), to determine the effect of these substances on enzyme activity.

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The amount of sugar produced after the enzymatic reaction was measured by high

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performance liquid chromatography (SHIMADZU LC-20A, Kyoto, Japan) with

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refractive index detector (RID). InertsilTM HPLC COLUM was used as analytical

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column using 75% acetonitrile and 25% double distilled water as the mobile phase.

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The conversion rate was measured as the ratio of the trehalose product to the initial

149

amount of maltose substrate. The amounts of trehalose, maltose, and glucose were

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determined using a trehalose reference standard (purity >99.5%; Sigma-Aldrich, St.

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Louis, MO). The enzyme activity unit was defined as the amount of enzyme that

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catalyzes the formation of 1 µmol trehalose per minute.

153 154

Crystallization and Data Collection. In order to obtain the crystal of TtTS, the

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purified protein was concentrated to 8–10 mg/mL by ultrafiltration (Millipore Amicon

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156

Ultra-15, USA). Crystals of native TtTS were obtained by sitting drop vapor diffusion

157

with crystallization screen kits at 293 K. After optimization, crystals were grown in

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hanging drops by mixing equal volumes of protein and reservoir solutions (0.2 M

159

magnesium formate dihydrate) at 293 K.

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For data collection, the crystal was flash-frozen in liquid nitrogen with 15–20%

161

(v/v) glycerol as a cryoprotectant, and all X-ray diffraction data sets were collected at

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100 K on beam line BL17U at SSRF (Shanghai, China) equipped with a

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MARMO-SAIC CCD 225 detector.

164

The X-ray collection data were integrated and scaled using the HKL-2000

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program suite.32 The native TtTS structure was resolved by molecular replacement

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using Phaser from the CCP4 suite of programs33 with TreS from M. smegmatis

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(Protein Data Bank (PDB) entry 3ZO9) as the search model. Refinement was

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performed using the PHENIX crystallography suite34 and the COOT interactive model

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building program.35 The inhibitor TriS was added to the model based on the Fo-Fc

170

density map of the ligand structure. The final model had Rwork = 0.1684 and Rfree =

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0.2088 based on a subset of 34,058 reflections.

172

Diffraction data collection and refinement statistics are listed in Table 1. The final

173

model was checked and validated using PROCHECK,36 QMEAN,37 and ProQ38 model

174

quality assessment tools, which indicated that the model was of good quality.

175

Structure graphics were illustrated with the PyMOL molecular visualization system.39

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The atomic coordinates and structural factors of TtTS have been deposited in the PDB

177

with accession code PDB 5X7U.

178 179

Analysis of TreS 3D Structures. The polar surface areas of TreSs were calculated

180

using the GetArea program (http://curie.utmb.edu/getarea.html),40 whereas the

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intramolecular

interactions

were

identified

using

the

PIC

server

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(http://pic.mbu.iisc.ernet.in/.41 The cavity volume of TreSs was calculated using

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Cavity 3.0 (http://www.caver.cz/).42

184 185

Computational Prediction of TreS Thermostability. The root mean square

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deviation (RMSD) values of TreSs, which have a negative correlation with the

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thermostability,43 were calculated using the g_rms software of the Gromacs 4.5

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package after Molecular Dynamics (MD) simulations at 500 K for 5 ns. The B-factor

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values, i.e., atomic displacement parameters, of amino acids were calculated using the

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B-FITTER program.44 In this study, the 3D structures of TreSs were subjected to 10

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ns MD simulations at 300 K, respectively, for calculating the B-factor values.

192 193

RESULTS

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Expression, Purification, and Evaluation of the Biochemical Properties of

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TreS. The treS gene from T. terrenum was cloned into the pET-21b expression vector

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to generate pET-21b-treS and transformed to E. coli BL21 (DE3). The TtTS (as a 6×

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His-tagged fusion protein) overexpressed in E. coli BL21 (DE3) was purified by using

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Ni-NTA, Source-Q, and Superdex 200 columns. Purified TtTS was analyzed by

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SDS-PAGE after Superdex 200 purification.

200

The temperature-dependency activities of TtTS on the conversion of maltose to

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trehalose were determined. The enzyme showed maximum activity at 45 °C (Figure

202

1A) and retained 80% of its initial activity after heat treatment at 70 °C for 30 min

203

(Figure 1B). The pH-dependency activities of TtTS were assayed, and the enzyme

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showed maximum activity at pH 7.5 (Figure 1C). As shown in Figure 1D,

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approximately 70% of the maltose substrate (150 mM) was converted to trehalose

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206

after a 10-h reaction. The effects of metal ions and reagents were analyzed at 1 mM

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and 10 mM concentrations of a variety of substances (Table 2). When treated with 1

208

mM SDS, EDTA, Cu2+, or Ni2+, TreS activity was clearly inhibited. All other metals

209

and reagents showed no obvious effects at this concentration. At 10 mM, inhibitory

210

effects of these four reagents were more obvious. However, Zn2+, Fe2+, Ca2+, Mn2+,

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Mg2+, Ba2+, and K+ can improve the effects of TtTS.

212 213

Overall Structure of TtTS. The crystal structure of TtTS belongs to the I4122

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space group with the cell dimension a = 159.176 Å, b = 159.176 Å, c = 152.815 Å.

215

The final model was refined to 2.5 Å resolution, with Rwork and Rfree values of 17.6%

216

and 21.5%, respectively. The diffraction data and refinement statistics are shown in

217

Table 1. The crystal structure of TtTS showed that it was a monomer. Each TtTS

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monomer was composed of 544 amino acids. In the structure of TtTS, the electron

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densities of the N-terminus to Gln3 and the C-terminal Pro were not visible in the

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monomer, which may be because of the disordered structure. In the final model of

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TtTS, more than 96.51% of the residues were located in the favored regions of the

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Ramachandran plot and only 3.31% in the generous and allowed regions.

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TtTS belongs to glycoside hydrolase family GH13. Our structure from T. terrenum

224

shares the common organization of other GH13 members, consisting of three major

225

domains: A, B, and C (Figure 2A). Domain A is located in the N-terminus, which

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consists of the (β/α)8 barrel (TIM barrel) structure and harbors the active site. The

227

second structure of domain A contains β1-β3, β7-β11, α1-α2, and α4-α12. The

228

catalytic cleft consists of the triad Asp202-Glu244-Asp310 and various conserved

229

substrate-binding residues. Domain B (residues 103–178) is inserted between β3 and

230

α4 of the TIM barrel and contains one short α-helix (α3) and three antiparallel

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β-strands (β4, β5, and β6). This domain is connected to domain A with which it has

232

tight interactions consisting of hydrogen bonds and hydrophobic contacts that are

233

mainly contributed by the loop-rich subdomain S7 (residues 329–353). S7 is located

234

between α9 and α10. Domain C (residues 453–549), located in the C-terminus,

235

comprises a seven-stranded antiparallel β-sandwich (β13-β19). Finally, there are

236

additional long loops inserted in the domains between β3 and α3, β6 and α4, and β7

237

and α5.

238 239

Structure of the Active Site. Structural comparison of TtTS with Deinococcus

240

radiodurans TreS (DrTS) and M. smegmatis TreS (MsTS) revealed that our TtTS

241

structure resembles those of many other GH13 members;29 for example, the conserved

242

active-site residues form similar extensive interaction networks with the catalytic triad

243

(Asp202-Glu244-Asp310). In particular, the superposition of TtTS with DrTS

244

demonstrates that the active site architecture is the same. The nucleophile Asp202

245

forms a salt bridge with Arg200, which also forms a salt bridge with Glu100 (replaced

246

by Arg101 in DrTS), whereas the general acid/base Glu244 forms a hydrogen bond

247

with the carbonyl backbone of Ala210 (Figure 3A,B).

248 249

Ion Binding Sites and mutagenesis analysis. In DrTS and MsTS, one Mg2+ and

250

one Ca2+ ion were identified in the structure,29,30 whereas four metal ion-binding sites

251

could be observed in the TtTS structure (Figure 4A). Among these, two metal

252

ion-binding sites are located in positions similar to those of DrTS and MsTS. Hence,

253

the same divalent cations were modeled into the electron density. Specifically, Site1 is

254

coordinated by the side chains of Asp23, Asn25, Asp27, Asp31 and the main-chain O

255

atom of Ser29, which was replaced by Lys30 in DrTS (Figure 4B). These amino acids

11



256

form part of the consensus sequence DX(S)NX(N)DGX(S)GD, which is also found in

257

some other GH13 family members.45,46 In turn, Site2 is coordinated by the side chains

258

of Asn104, Asp172, Glu209, and the backbone O atoms of Tyr206 and Leu207. These

259

surrounding amino acids are conserved within DrTS (Figure 4C). A similar site

260

containing the conserved Asp and Asn residues is also found in many α-amylases.47

261

Of the two additional metal ion-binding sites in TtTS (Site3 and Site4), Site3 is

262

coordinated by the side chains of Arg283, Arg291, Thr287, Arg471, and one water

263

molecule (Figure 4D). Site4 is coordinated by the side chains of Arg355, Ser352,

264

Arg354, and His534 (Figure 4E). These two metal ion-binding sites are not observed

265

in other TreS structures. We therefore considered that the additional two metal

266

ion-binding sites may be associated with protein stability.

267

To confirm the structure analysis result, the amino acids around Site3 and Site4

268

were mutated, generating three mutant combinations (Site3: R283G/Y287R/R291G,

269

Site4: H534Y, and Site3-4: R283G/Y287R/R291G/H534Y). The thermal stability of

270

each mutant was then measured four times (Figure S1). The results showed that Site3

271

mutants (R283G/Y287R/R291G), Site4 mutants (H534Y) and Site3-4 mutants

272

(R283G/Y287R/R291G/H534Y) retained 36%, 50%, and 25% activity, respectively,

273

at 70 °C for 30 min. The thermal stability of all mutants was reduced compared to that

274

of the native enzyme.

275 276

Comparison with Homologous Structures. A structure alignment was next

277

carried out using the DALI program48 against PDB entries. The similarity results

278

showed that the scaffold of TtTS is highly conserved among all known GH13 family

279

structures. DrTS (PDB: 4tvu-A, Z-score 63.2)28 has the most similarity to TtTS,

280

followed by MsTS (PDB: 3zo9-A, Z-score 54.3)29 and TreS from Mycobacterium

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tuberculosis (MtTS; PDB: 4lxf-A, Z-score 48.0).30

282

The structure and characteristics of mesophilic (DrTS) and thermophilic (TtTS)

283

TreSs were calculated and compared, as shown in Supplementary Table S2. In

284

comparison with DrTS, TtTS has more polar (charged) amino acids distributed on the

285

surface of its structure and more aromatic amino acids buried within. The polar

286

surface area, total surface area, and cavity volume of TtTS calculated using GetArea

287

and Cavity 3.0 were 8844.73 Å2, 20729.78 Å2, and 7377.49 Å3, larger than those

288

(7714.37 Å2, 19864.15 Å2, and 7138.84 Å3) of DrTS, respectively, whereas the

289

number of surface amino acids of TtTS was less than that of DrTS. As calculated by

290

PIC, the numbers of hydrophobic interactions, hydrogen bonds between main

291

chain-side chain and side chain-side chain, ionic interactions, and aromatic-aromatic

292

interactions of TtTS were larger than those of DrTS.

293

The Cα superposition of the three different species of TreS yielded RMSD values

294

of 1.0, 1.5, and 1.6 Å, respectively. The (β/α) 8 barrel was the most overlapped part in

295

the structure. In domain A, the most distinguishing parts were between β9 and α10

296

containing S7, α9, and loops between β9 and α9 (Figure 5A). These divergent parts

297

are located on the top of the active site and may play roles in modulating its shape and

298

accessibility. The most divergent parts were Domains B and C. In Domain B, the most

299

diverse segments were the regions β4, β5, β6, and the loops between β4 and β5

300

(Figure 5B), which are located on the top of the active site and have interactions with

301

S7 of Domain A. The function of Domain B may be to modulate substrate access and

302

binding. The other small distinguishing part is between α3 and β4 (Figure 5B), which

303

may be associated with protein stability and will be discussed later in further detail.

304

Domain C displayed a distinct lack of significant sequence similarity.

305

Superposition of Domain C revealed that β13, β14, and β15 are tightly packed with

13



306

the (β/α) 8 barrel and exhibit sequence similarity. The most distinguishing parts of

307

Domain C were β15, β16, β17, and β18, which anticipate the functional diversity

308

(Figure 5C). For example, a number of GH13 members function as a

309

carbohydrate-binding module.43 In comparison, Domain C also mediates the oligomer

310

state in GH13 members. Most GH13 members are monomeric; however, DrTS is

311

dimeric, and MsTS forms a tetramer.30 The sequence diversity of Domain C accounts

312

for the hydrogen bonds and hydrophobic contacts observed in the oligomer state

313

interfaces. In our structure, TtTS was monomeric.

314

Thermophilic Analysis by MD Simulation. MD simulation at high temperature

315

provides an insight into protein unfolding, which is negatively related to its

316

thermostability.49 In order to calculate the RMSD values of DrTS and TtTS, their 3D

317

structures were subjected to MD simulations at 500 K. As shown in Figure 6A, the

318

RMSD values of TtTS after equilibration were lower than those of DrTS, indicating

319

that TtTS was more rigid than DrTS. We also calculated the B-factor values of DrTS

320

and TtTS using Gromacs 4.5 (Figure 6B). As shown in Fig 6C, two contiguous

321

regions (Asp4-Val129 and Tyr373-Gln450) in TtTS were smaller than the

322

corresponding regions in MsTS. Moreover, analysis results of the 3D structure of

323

TtTS using PYMOL and PIC indicated that there was a close interaction between

324

regions Asp4-Val129 and Tyr373-Gln450 of TtTS, which suggests that these two

325

regions may significantly correlate with its thermostability.

326 327

DISCUSSION

328

TtTS, identified from T. terrenum, represents a new kind of thermophilic TreS.

329

TtTS exhibited maximum activity at 45 °C and retained 80% enzyme activity after

330

heat treatment at 70 °C for 30 min. Furthermore, it could convert approximately 70%

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331

of the maltose substrate (150 mM) into trehalose after a 10-h reaction with byproduct

332

(glucose). Comparison of TtTS with TreSs from other sources demonstrated that it

333

had obvious thermal stability. However, TtTS differed from other thermophilic TreS

334

enzymes, such as that from Thermus thermophilus, which has greater molecular mass

335

owing to an extra C-terminal region.50 Therefore, TtTS represents a new kind of TreS

336

with low molecular mass.51

337

In a number of GH13 enzymes, a similar architecture displays conserved residues

338

in the active site. Sequence-based alignment of TtTS with previously determined

339

structures of TreSs showed that the triad catalytic residues Asp202-Glu244-Asp310

340

are highly conserved (Figure 7). In this regard, TtTS appears to have a similar

341

catalytic mechanism as other TreSs. Specifically, the nucleophilic reagent Asp202

342

attacks the sugar anomeric center of maltose in an acid-catalyzed process by forming

343

a covalent β-glucose-enzyme intermediate. Next, glucose is released and retained

344

within the active site; the 1-hydroxyl then attacks the anomeric center of the glycosyl

345

enzyme. Glu244 plays the role of an acid/base catalyst and attacks the enzyme

346

intermediate to form trehalose and regenerate the free enzyme. Asp310 functions in

347

substrate binding.

348

TtTS also has the typical glycoside hydrolase family GH13 structure. However,

349

TtTS has thermophilic structural characteristics. TtTS has more polar (charged) amino

350

acids than mesophilic TreSs, which may lead to the formation of more hydrogen and

351

ionic bonds. The hydrophobic interactions and aromatic-aromatic interactions may in

352

turn lend the protein interior greater stability. In contrast, many GH13 members have

353

been reported to contain two metal ions binding sites; however, four potential metal

354

ion-binding sites were observed in the structure of TtTS. Metal ions may serve to

355

maintain structural integrity.52 Together, these factors likely result in rendering TtTS

15



356

more stable than other mesophilic TreSs. In addition, MD simulation further

357

suggested that two regions (Asp4-Val129 and Tyr373-Gln450) may be highly

358

significantly correlated to the thermostability of TtTS.

359

Trehalose has important applications in the food, pharmaceutical, and cosmetic

360

industries, and efficient production methods can reduce costs. Thermophilic enzymes

361

have many advantages for industrial application. However, few thermophilic enzymes

362

are currently utilized for production, mainly because of the lengthy reaction time and

363

possible generation of byproduct glucose. The detailed thermophilic enzyme structure

364

may therefore supply guidance for further protein engineering toward the design of

365

efficient enzymes. In this study, we showed the structure of the novel thermophilic

366

TreS, TtTS, and illuminated the mechanism of its thermostability. Further engineering

367

of this enzyme may in turn improve its catalytic efficiency to facilitate the production

368

of trehalose at low cost.

369

Abbreviations: DrTS, Deinococcus radiodurans TreS; GH13, glycoside hydrolase

370

family 13; MsTS, Mycobacterium smegmatis TreS; PDB, protein data bank;

371

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TreS,

372

trehalose synthase; TriS, Tris (Hydroxymethyl) aminomethane; TtTS, trehalose

373

synthase from Thermobaculum terrenum

374

Acknowledgements

375

We thank the staff at beam line BL17U at the Shanghai Synchrotron Radiation

376

Facility for support with data collection. We thank Professor Gu’s Lab at the State

377

Key Laboratory of Microbial Technology, Shandong University for protein

378

purification and crystallization. This work was supported by the National Natural

379

Science Foundation of China (31401626) and Science Foundation of ShanDong

380

Province (ZR2014CQ039).

16


Page 16 of 33

Page 17 of 33


381

Supporting Information

382

Table S1. Primers used to mutate TtTS around the metal binding sites of Sites 3 and

383

Sites 4

384

Table S2. The structure and characteristics difference of DrTs and TtTs

385

Figure S1. Temperature stability of three mutant combinations of TtTS. Enzyme

386

assays for all mutants were performed as for wild type TtTS. Error bars indicate the

387

standard deviation. The results figure was generated using OriginPro 8. (A) Site3

388

(R283G/Y287R/R291G) mutant thermal stability. (B) Site4 (H534Y) mutant thermal

389

stability. (C) Site3-4 (R283G/Y287R/R291G/H534Y) mutant thermal stability.

390 391

Author Information

392

Correspondig Author

393

*J.S. E_mail: [email protected]. Phone: +86-0531-88363022

394

Author Contributions

395

Q.W. and D.R. contributed equally to this paper. All authors have given approval to

396

the final version of the manuscript. J.S. designed the experiment and drafted the

397

manuscript. Q.W. and D.R. performed most of experiments and data analysis. M.W.

398

revised the manusript. F.W. conducted the crystal data collection.

399

Notes

400

The authors declare no competing financial interest.

401

References

402 403 404 405 406 407 408

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17



409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452

Escherichia coli to cold and is essential for viability at low temperatures. P NATL ACAD SCI USA 2002, 99, (15), 9727-9732. 4. Mahmud, S. A.; Nagahisa, K.; Hirasawa, T.; Yoshikawa, K.; Ashitani, K.; Shimizu, H., Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae. YEAST 2009, 26, (1), 17-30. 5. Al-Bader, N.; Vanier, G.; Liu, H.; Gravelat, F. N.; Urb, M.; Hoareau, M. Q.; Campoli, P.; Chabot, J.; Filler, S. G.; Sheppard, D. C., Role of trehalose biosynthesis in Aspergillus fumigatus development, stress response, and virulence. INFECT IMMUN 2010, 78, (7), 3007-3018. 6. Hunter, R. L.; Armitige, L.; Jagannath, C.; Actor, J. K., TB Research at UT-Houston – A review of cord factor: new approaches to drugs, vaccines and the pathogenesis of tuberculosis. TUBERCULOSIS 2009, 89, (12), S18-S25. 7. Kalscheuer, R.; Weinrick, B.; Veeraraghavan, U.; Besra, G. S.; Jr, J. W., Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2010, 107, (50), 21761-21766. 8. Roser, B., Trehalose, a new approach to premium dried foods. TRENDS FOOD SCI TECH 1991, 2, (7), 166-169. 9. Maruta, K.; Nakada, T.; Kubota, M.; Chaen, H.; Sugimoto, T.; Kurimoto, M.; Tsujisaka, Y., Formation of trehalose from maltooligosaccharides by a novel enzymatic system. BIOSCI BIOTECH BIOCH 1995, 59, (10), 1829-1834. 10. Paiva, C. L.; Panek, A. D., Biotechnological applications of the disaccharide trehalose. BIOTECHNOL ANNU REV 1996, 2, (8), 293-314. 11. Guo, N.; Puhlev, I. D.; Mansbridge, J.; Levine, F., Trehalose expression confers desiccation tolerance on human cells. NAT BIOTECHNOL 2000, 18, (2), 168171. 12. Styrvold, O. B.; Strøm, A. R., Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase. J BACTERIOL 1991, 173, (3), 1187-1192. 13. Giaever, H. M.; Styrvold, O. B.; Kaasen, I.; Strøm, A. R., Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli. J BACTERIOL 1988, 170, (6), 2841-2849. 14. Pan, Y. T.; Carroll, J. D.; Elbein, A. D., Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme. Eur J Biochem 2002, 269, (24), 6091-6100. 15. Edavana, V. K.; Pastuszak, I.; Carroll, J. D.; Thampi, P.; Abraham, E. C.; Elbein, A. D., Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis. Arch Biochem Biophys 2004, 426, (2), 250-257. 16. Nakao, M.; Harada, M.; Kodama, Y.; Nakayama, T.; Shibano, Y.; Amachi, T., Purification and properties of a novel enzyme, maltooligosyl trehalose synthase, from Arthrobacter sp. Q36. BIOSCI BIOTECH BIOCH 1995, 59, (12), 2210-2214. 17. Nakada, T.; Maruta, K.; Mitsuzumi, H.; Kubota, M.; Chaen, H.; Sugimoto, T.; Kurimoto, M.; Tsujisaka, Y., Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp. Q36. BIOSCI BIOTECH BIOCH 1995, 59, (12), 2215-2218. 18. Nishimoto, T.; Nakano, M.; Nakada, T.; Chaen, H.; Fukuda, S.; Sugimoto, T.; Kurimoto, M.; Tsujisaka, Y., Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48. BIOSCI BIOTECH BIOCH 1996, 60, (4), 640-644.

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19. Elbein, A. D.; Pan, Y. T.; Pastuszak, I.; Carroll, D., New insights on trehalose: a multifunctional molecule. GLYCOBIOLOGY 2003, 13, (4), 17R-27R. 20. Pan, Y. T.; Koroth, E. V.; Jourdian, W. J.; Edmondson, R.; Carroll, J. D.; Pastuszak, I.; Elbein, A. D., Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme. Eur J Biochem 2004, 271, (21), 4259-4269. 21. Lee, J.; Lee, K.; Kim, C.; Lee, S.; Kim, G.; Park, Y.; Chung, S., Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose. APPL MICROBIOL BIOT 2005, 68, (2), 213 - 219. 22. Chen, Y.; Lee, G.; Shaw, J., Gene Cloning, Expression, and Biochemical Characterization of a Recombinant Trehalose Synthase from Picrophilus torridusin Escherichia coli. J AGR FOOD CHEM 2006, 54, (19), 7098-7104. 23. Wu, X.; Ding, H.; Ming, Y.; Yu, Q., Gene cloning, expression, and characterization of a novel trehalose synthase from Arthrobacter aurescens. APPL MICROBIOL BIOT 2009, 83, (3), 477-482. 24. Yue, M.; Wu, X. L.; Gong, W. N.; Ding, H. B., Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei. MICROB CELL FACT 2009, 8, (1), 1-7. 25. Zhu, Y.; Wei, D.; Zhang, J.; Wang, Y.; Xu, H.; Xing, L.; Li, M., Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber. EXTREMOPHILES 2010, 14, (1), 1-8. 26. Wang, J. H.; Tsai, M. Y.; Chen, J. J.; Lee, G. C.; Shaw, J. F., Role of the C-Terminal Domain of Thermus thermophilus Trehalose Synthase in the Thermophilicity, Thermostability, and Efficient Production of Trehalose. J AGR FOOD CHEM 2007, 55, (9), 3435-3443.27. Nishimoto, T.; Nakada, T.; Chaen, H.; Fukuda, S.; Sugimoto, T.; Kurimoto, M.; Tsujisaka, Y., Purification and Characterization of a Thermostable Trehalose Synthase from Thermus aquaticus. BIOSCI BIOTECH BIOCH 1996, 60, (5), 835-839. 28. Wang, Y. L.; Sih-Yao, C.; Lin, Y. T.; Yu-Chiao, H.; Guan-Chiun, L.; Shwu-Huey, L., Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization. Acta Crystallographica 2014, 70, (Pt 12), 3144-3154. 29. Caner, S.; Nguyen, N.; Aguda, A.; Zhang, R.; Pan, Y. T.; Withers, S. G.; Brayer, G. D., The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode. GLYCOBIOLOGY 2013, 23, (9), 1075-1083. 30. Rana Roy, V. U. A. K., Synthesis of α-Glucan in Mycobacteria Involves a Hetero-octameric Complex of Trehalose Synthase TreS and Maltokinase Pep2. ACS CHEM BIOL 2013, 8, (10), 2245-2255. 31. Zhang, R.; Pan, Y. T.; He, S.; Lam, M.; Brayer, G. D.; Elbein, A. D.; Withers, S. G., Mechanistic analysis of trehalose synthase from Mycobacterium smegmatis. J BIOL CHEM 2011, 286, (41), 35601. 32. Otwinowski, Z.; Minor, W., Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 1997, 276, (97), 307-326. 33. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; Mccoy, A., Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 2011, 67, (Pt 4), 235-242. 34. Adams, P. D.; Grossekunstleve, R. W.; Hung, L. W.; Ioerger, T. R.; Mccoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C., PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 2002, 58, (Pt 11), 1948-1954.

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497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539

35. Emsley, P.; Cowtan, K., Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004, 60, (Pt 12 Pt 1), 2126-2132. 36. Laskowski, R. A.; Macarthur, M. A.; Moss, D. S.; Thornton, J. M., 20.19. (1993). PROCHECK a program to check the stereochemical quality of protein structures. J. App. Cryst 1993, 26,283-291. 37. Benkert, P.; Tosatto, S. C. E.; Schomburg, D., QMEAN: A comprehensive scoring function for model quality assessment. PROTEINS 2008, 71, (1), 261-277. 38. Cristobal, S.; Zemla, A.; Fischer, D.; Rychlewski, L.; Elofsson, A., A study of quality measures for protein threading models. BMC BIOINFORMATICS 2001, 2, (1), 5. 39. Lill, M. A.; Danielson, M. L., Computer-aided drug design platform using PyMOL. J COMPUT AID MOL DES 2011, 25, (1), 13-19. 40. Fraczkiewicz, R.; Braun, W., Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J COMPUT CHEM 1998, 19, (3), 319-333. 41. Tina, K. G.; Bhadra, R.; Srinivasan, N., PIC: Protein Interactions Calculator. NUCLEIC ACIDS RES 2007, 35, (Web Server issue), 473-476. 42. Eva, C.; Antonin, P.; Petr, B.; Ondrej, S.; Jan, B.; Barbora, K.; Artur, G.; Vilem, S.; Martin, K.; Petr, M.; Lada, B.; Jiri, S.; Jiri, D., CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLOS COMPUT BIOL 2012, 8, (10). 43. Badieyan, S.; Bevan, D. R.; Zhang, C. M., Study and design of stability in GH5 cellulases. BIOTECHNOL BIOENG 2012, 109, (1), 31-44. 44. Reetz, M. T.; Carballeira, J. D., Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. NAT PROTOC 2007, 2, (4), 891-903. 45. Ravaud, S.; Robert, X.; Watzlawick, H.; Haser, R.; Mattes, R.; Aghajari, N., Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization. J BIOL CHEM 2007, 282, (38), 28126-28136. 46. Yamamoto, K.; Miyake, H.; Kusunoki, M.; Osaki, S., Crystal structures of isomaltase from Saccharomyces

cerevisiae and in complex with its competitive inhibitor maltose. FEBS J 2010, 277,

(20), 4205-4214. 47. Janeček, Š.; Svensson, B.; Macgregor, E. A., α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. CELL MOL LIFE SCI 2014, 71, (7), 1149-1170. 48. Lefebvre, J. L.; Ang, K. K., In Reply to Dr. Christiansen et al. INT J RADIAT ONCOL 2009, 75, (2), 633-634. 49. Purmonen, M.; Valjakka, J.; Takkinen, K.; Laitinen, T.; Rouvinen, J., Molecular dynamics studies on the thermostability of family 11 xylanases. PROTEIN ENG DES SEL 2007, 20, (11), 551-559. 50. Silva, Z.; Alarico, S.; Nobre, A.; Horlacher, R.; Marugg, J.; Boos, W.; Mingote, A. I.; Costa, M. S. D., Osmotic Adaptation of Thermus thermophilus RQ-1: Lesson from a Mutant Deficient in Synthesis of Trehalose. J BACTERIOL 2003, 185, (20), 5943-5952. 51. Kiss, H.; Cleland, D.; Lapidus, A.; Lucas, S.; Rio, T. G. D.; Nolan, M.; Tice, H.; Han, C.; Goodwin, L.; Pitluck, S., Complete genome sequence of ‘Thermobaculum terrenum’ type strain (YNP1T). STAND GENOMIC SCI 2010, 3, (2), 153-162. 52. Kobayashi, M.; Hondoh, H.; Mori, H.; Saburi, W.; Okuyama, M., Calcium ion-dependent increase in thermostability of dextran glucosidase from Streptococcus mutans. BIOSCI BIOTECH BIOCH 2011, 75, (8), 1557-1563.

20


Page 20 of 33

Page 21 of 33


540

Figure 1. Enzyme characteristic of TtTS: (A) enzyme activities at various

541

temperatures (30–85 °C) were assayed; (B) enzyme thermal stabilities were measured

542

by incubating at various temperatures (40–80 °C), and the residual activities were

543

assayed at 40 °C; (C) enzyme activities at various pH (pH4.0-11.0); (D) conversion

544

yield of maltose to trehalose by TtTS.

545 546

Figure 2. Overall structure of TtTS: (A) the complete structures of domains A, B, and

547

C are shown in magenta, green, and yellow, respectively, in a cartoon model. The

548

connection of S7 to domains A and B is shown in blue; (B) the secondary structures of

549

the TtTS monomer are depicted in a rainbow-colored cartoon model.

550 551

Figure 3. Structure of TtTS-TriS complex: (A) the difference in the electron density

552

map (FO-FC) calculated at 2.5 Å resolution using phase from the final model with TriS

553

and contoured at 2.0σ reveals the existence of TriS with clear electron density within

554

the molecule; (B) comparison of TtTS and MsTS structures. Yellow ribbon represents

555

the TtTS structure; cyan ribbon represents the MsTS structure. TriS is shown as

556

colored sticks. In the TtTS structure, triad catalytic sites (Asp202-Glu244-Asp310)

557

are indicated by an asterisk and other catalytic network amino acids (Arg307, Asn308,

558

Arg311, Arg343, Asp388, Arg389, Ala203, Asp62, and Glu311) are shown as yellow

559

sticks. Homologous amino acids in MsTS are shown as blue sticks.

560 561

Figure 4. The metal-binding site in TtTs (magenta ball = metal binding site; amino

562

acids in TtTS are shown as green sticks; amino acids in DrTS are shown as white

563

sticks): (A) the difference in the electron density map (FO-FC) contoured at 2.0σ

564

reveals the existence of four metal sites (m); (B) coordinated amino acids in

21



565

metal-binding Site1; (C) coordinated amino acids in metal-binding Site2; (D)

566

coordinated amino acids in metal-binding Site3; (E) coordinated amino acids in

567

metal-binding Site4.

568 569

Figure 5. Structure superpositions of TtTS (green) with DrTS (cyan; PDB entry 4tvu)

570

and MsTS (magenta; PDB entry 3zo9): (A) the most distinguishing parts in Domain A;

571

(B) the most distinguishing parts in Domain B; (C) the most distinguishing parts in

572

Domain C.

573 574

Figure 6. MD analysis results: (A) simulation curves of RMSD values of TtTS (red)

575

and DrTS (blue) after MD simulations at 500 K for 5 ns; (B) B-factors of amino acids

576

of DrTS and TtTS are marked in blue solid and red dashed lines, respectively; (C)

577

B-factor difference between DrTS and TtTS.

578 579

Figure 7. Multiple sequence alignment of TtTS with DrTS (PDB entry 4tvu), MsTS

580

(PDB entry 3zo9), and MtTS (PDB entry 4lxf). Highly conserved triad catalytic sites

581

are labeled with red asterisks.

22


Page 22 of 33

Page 23 of 33


Table 1.

Data collection and refinement statistics TtTS-TriS (PDB: 5X7U )

Data collection 2.50

Resolution(Å)

(50 - 2.50) * I4122

Space group Cell dimensions a, b, c (Å)

159.176, 159.176, 152.815

α, β, γ (°)

90, 90, 90 14.3(14.7)

Redundancy

34058(3319)

Unique reflections (outer shell)

63.6 (20.0)

I/σ (outer shell)

0.025(0.992)

Rsym (outer shell) Refinement

2.50

Resolution (Å)

34034

Reflections used in refinement No. atoms

4549

Protein

7

Ligand/ion

237

Water Ramachandran (%)

96.51

Most favored

23



3.31

Generously allowed

0.18

Disallowed

0.1760/0.2148

R-work/ R-free R.m.s deviations

0.010

Bond lengths (Å)

1.031

Bond angles (º) *Highest resolution shell is shown in parenthesis.

24


Page 24 of 33

Page 25 of 33


Table 2 Effects of metal ions and reagents on the activity of TtTS Reagent

Relative Activity (%) 1mM

10mM

none

100±3.11

100±3.23

ZnSO4

101±3.13

FeSO4

Reagent

Relative Activity (%) 1mM

10mM

BaCl2

105±2.52

103±1.99

119±2.74

KCl

104±7.60

102±11.05

103±4.06

109±5.12

CaCl2

101±9.24

108±3.68

CuSO4

66±2.26

59±6.86

MnCl2

100±9.46

108±1.74

SDS

23±4.80

0±0.00

NiCl2

34±2.24

25±3.48

EDTA

54±5.67

0±0.00

MgCl2

100±0.97

108±8.59

25



Figure 1. Enzyme characteristic of TtTS: (A) enzyme activities at various temperatures (30–85 °C) were assayed; (B) enzyme thermal stabilities were measured by incubating at various temperatures (40–80 °C), and the residual activities were assayed at 40 °C; (C) enzyme activities at various pH (pH4.0-11.0); (D) conversion yield of maltose to trehalose by TtTS. 42x42mm (600 x 600 DPI)


Page 26 of 33

Page 27 of 33


Figure 2. Overall structure of TtTS: (A) the complete structures of domains A, B, and C are shown in magenta, green, and yellow, respectively, in a cartoon model. The connection of S7 to domains A and B is shown in blue; (B) the secondary structures of the TtTS monomer are depicted in a rainbow-colored cartoon model. 127x84mm (300 x 300 DPI)



Figure 3. Structure of TtTS-TriS complex: (A) the difference in the electron density map (FO-FC) calculated at 2.5 Å resolution using phase from the final model with TriS and contoured at 2.0σ reveals the existence of TriS with clear electron density within the molecule; (B) comparison of TtTS and MsTS structures. Yellow ribbon represents the TtTS structure; cyan ribbon represents the MsTS structure. TriS is shown as colored sticks. In the TtTS structure, triad catalytic sites (Asp202-Glu244-Asp310) are indicated by an asterisk and other catalytic network amino acids (Arg307, Asn308, Arg311, Arg343, Asp388, Arg389, Ala203, Asp62, and Glu311) are shown as yellow sticks. Homologous amino acids in MsTS are shown as blue sticks. 127x84mm (300 x 300 DPI)


Page 28 of 33

Page 29 of 33


Figure 4. The metal-binding site in TtTs (magenta ball = metal binding site; amino acids in TtTS are shown as green sticks; amino acids in DrTS are shown as white sticks): (A) the difference in the electron density map (FO-FC) contoured at 2.0σ reveals the existence of four metal sites (m); (B) coordinated amino acids in metal-binding Site1; (C) coordinated amino acids in metal-binding Site2; (D) coordinated amino acids in metal-binding Site3; (E) coordinated amino acids in metal-binding Site4. 152x211mm (300 x 300 DPI)



Figure 5. Structure superpositions of TtTS (green) with DrTS (cyan; PDB entry 4tvu) and MsTS (magenta; PDB entry 3zo9): (A) the most distinguishing parts in Domain A; (B) the most distinguishing parts in Domain B; (C) the most distinguishing parts in Domain C. 169x169mm (300 x 300 DPI)


Page 30 of 33

Page 31 of 33


Figure 6. MD analysis results: (A) simulation curves of RMSD values of TtTS (red) and DrTS (blue) after MD simulations at 500 K for 5 ns; (B) B-factors of amino acids of DrTS and TtTS are marked in blue solid and red dashed lines, respectively; (C) B-factor difference between DrTS and TtTS. 169x169mm (300 x 300 DPI)



Figure 7. Multiple sequence alignment of TtTS with DrTS (PDB entry 4tvu), MsTS (PDB entry 3zo9), and MtTS (PDB entry 4lxf). Highly conserved triad catalytic sites are labeled with red asterisks. 84x84mm (300 x 300 DPI)


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Table of Contents 85x47mm (300 x 300 DPI)


Structural Characteristics and Function of a New Kind of Thermostable

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