Maltooligosyltrehalose Synthase Research Articles

Analysis of the trehalose synthesis pathway of Physarum polycehalum

Desiccation is a severe survival problem for organisms. We have been studying the desiccation tolerance mechanisms in the true slime mold Physarum polycephalum. We measured the trehalose content of P. polycephalum vegetative cells (plasmodia) and drought cells (sclerotia). Surprisingly, we found that the content in sclerotia was about 473-fold greater than in the plasmodia. We then examined trehalose metabolism-related genes via RNAseq, and consequently found that trehalose 6-phosphate phosphorylase (T6pp) expression levels increased following desiccation. Next, we cloned and expressed the genes for T6pp, trehalose 6-phosphate synthase/phosphatase (Tps/Tpp), maltooligosyltrehalose trehalohydrolase (TreZ), and maltooligosyltrehalose synthase (TreY) in E. coli. Incidentally, TreY and TreZ clones have been reported in several prokaryotes, but not in eukaryotes. This report in P. polycephalum is the first evidence of their presence in a eukaryote species. Recombinant T6pp, TreY, and TreZ were purified and confirmed to be active. Our results showed that these enzymes catalyze reactions related to trehalose production, and their reaction kinetics follow the Michaelis-Menten equation.The t6pp mRNA levels of the sclerotia were about 15-fold higher than in the plasmodia. In contrast, the expression levels of TreZ and TreY showed no significant change between the sclerotia and plasmodia. Thus, T6pp is probably related to desiccation tolerance, whereas the contribution of TreY and TreZ is insufficient to account for the considerable accumulation of trehalose in sclerotia.

In vitro assembly of the trehalose bi-enzyme complex with artificial scaffold protein.

Introduction: Trehalose is a significant rare sugar known for its stable properties and ability to protect biomolecules from environmental factors. Methods: In this study, we present a novel approach utilizing a scaffold protein-mediated assembly method for the formation of a trehalose bi-enzyme complex. This complex consists of maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase), which work in tandem to catalyze the substrate and enhance the overall catalytic efficiency. Utilizing the specific interaction between cohesin and dockerin, this study presents the implementation of an assembly, an analysis of its efficiency, and an exploration of strategies to enhance enzyme utilization through the construction of a bi-enzyme complex under optimal conditions in vitro. Results and Discussion: The bi-enzyme complex demonstrated a trehalose production level 1.5 times higher than that of the free enzyme mixture at 40h, with a sustained upward trend. Compared to free enzyme mixtures, the adoption of a scaffold protein-mediated bi-enzyme complex may improve cascade reactions and catalytic effects, thus presenting promising prospects.

Trehalose Production Using Three Extracellular Enzymes Produced via One-Step Fermentation of an Engineered Bacillus subtilis Strain.

At present, the double-enzyme catalyzed method using maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) is the mainstream technology for industrial trehalose production. However, MTSase and MTHase are prepared mainly using the heterologous expression in the engineered Escherichia coli strains so far. In this study, we first proved that the addition of 3 U/g neutral pullulanase PulA could enhance the trehalose conversion rate by 2.46 times in the double-enzyme catalyzed system. Then, a CBM68 domain was used to successfully assist the secretory expression of MTSase and MTHase from Arthrobacter ramosus S34 in Bacillus subtilis SCK6. At the basis, an engineered strain B. subtilis PSH02 (amyE::pulA/pHT43-C68-ARS/pMC68-ARH), which co-expressed MTSase, MTHase, and PulA, was constructed. After the 24 h fermentation of B. subtilis PSH02, the optimum ratio of the extracellular multi-enzymes was obtained to make the highest trehalose conversion rate of 80% from 100 g/L maltodextrin. The high passage stability and multi-enzyme preservation stability made B. subtilis PSH02 an excellent industrial production strain. Moreover, trehalose production using these extracellular enzymes produced via the one-step fermentation of B. subtilis PSH02 would greatly simplify the procedure for multi-enzyme preparation and be expected to reduce production costs.

Permeabilized whole cells containing co-expressed cyclomaltodextrinase and maltooligosyltrehalose synthase facilitate the synthesis of nonreducing maltoheptaose (N-G7) from β-cyclodextrin.

Maltodextrin is an important bulk ingredient in food and other industries; however, drawbacks such as uneven polymerization and high reducibility limit its utilization. Nonreducing maltoheptaose (N-G7) is a good substitute for maltodextrin owing to its single degree of polymerization and nonreducing properties. Herein, in vitro cell factory biotransformation of β-cyclodextrin (β-CD) to N-G7 is demonstrated using coexpressed cyclomaltodextrinase (CDase, EC 3.2.1.54) and maltooligosyltrehalose synthase (MTSase, EC 5.4.99.15). However, the cell membrane prevents β-CD from entering the cell owing to its large diameter. The amylase-deficient permeabilized host ΔycjM-ΔmalS-ΔlpxM is utilized for coexpression of recombinant CDase and MTSase. Deletion of lpxM effectively allows the entry of β-cyclodextrin into the cell, despite its large diameter, without requiring any relevant cell membrane permeability-promoting reagent. This results in a 28.44% increase in the efficiency of β-CD entry into the cell, thus enabling intracellular N-G7 synthesis without the extracellular secretion of recombinant CDase and MTSase. After reacting for 5.5 h, the highest purity of N-G7 (65.50%) is obtained. However, hydrolysis decreases the purity of N-G7 to 49.30%, thus resulting in a conversion rate of 40.16% for N-G7 when the reaction lasts 6 h. Precise control of reaction time is crucial for obtaining high-purity N-G7. Whole-cell catalysis avoids cell fragmentation and facilitates the creation of an eco-friendly, energy-efficient biotransformation system; thus, it is a promising approach for N-G7 synthesis. This article is protected by copyright. All rights reserved.

Physicochemical properties of a non-reducing maltoheptaose prepared by dual-enzyme cascade reaction from starch

The existence of a reducing end in the structure of maltodextrin can limit its applications as undesirable Maillard reaction would occur in some food processing steps. Consequently, a non-reducing maltoheptaose (N-G7) with a single degree of polymerisation was prepared through a cascade reaction of cyclodextrinase and maltooligosyltrehalose synthase, using β-cyclodextrin as substrate. The physicochemical properties of N-G7 were investigated. N-G7 exhibited low moisture absorption ability (8.91 and 18.02% at 43 and 81% relative humidity, respectively), excellent pH stability and thermostability (less than 10% N-G7 was hydrolysed between pH 4 and 10, even at 100°C), and a melting point higher than that of maltodextrin, as well as a typical gel-like behaviour. Most importantly, the results of Maillard reaction indicated that N-G7 was considered to be non-reducing, which suggested that it could be used in food processing where Maillard reaction should be avoided. Overall, the present work may provide important implications for the development and application of N-G7 in food products.

Deletion of α-amylase genes via CRISPR/Cas9 decreases the side effects of hydrolysis towards nonreducing maltoheptaose preparation

Maltodextrin is a significant ingredient extensively used in food industries, but its application is limited due to uneven polymerization and high reducibility. Hence, preparation of nonreducing maltodextrin with narrow distribution of DP is essential. A dual-enzyme cascade enzymatic reaction for preparing nonreducing maltoheptaose (N-G7) is feasible via recombinant cyclomaltodextrinase (EC 3.2.1.54, CDase) and maltooligosyltrehalose synthase (EC 5.4.99.15, MTSase) heterologously overexpressed in Escherichia coli BL21(DE3). However, during this process, N-G7 further was hydrolyzed into small molecule maltooligosaccharides by the amylases homologously expressed in the host. In this study, two α-amylase genes (ycjM and malS) were deleted to avoid host hydrolysis in N-G7 via CRISPR/Cas9 system. Gene deletion had a significant decrease on the yield of maltooligosaccharides of 5 and 6 glucose units. The defective strains ΔycjM and ΔycjM-ΔmalS increased the yield of N-G7 by 21.94% and 25.47%, correspondingly. Marginal impact was made in cell growth and protein secretion of the host. YcjM and MalS were overexpressed in ΔycjM-ΔmalS and the hydrolytic activity of YcjM was twice that of MalS. They constituted a complex multiple hydrolysis system for N-G7 with the Mal series enzymes involved in a maltose system in the host.

Prediction of trehalose-metabolic pathway and comparative analysis of KEGG, MetaCyc, and RAST databases based on complete genome of Variovorax sp. PAMC28711

BackgroundMetabolism including anabolism and catabolism is a prerequisite phenomenon for all living organisms. Anabolism refers to the synthesis of the entire compound needed by a species. Catabolism refers to the breakdown of molecules to obtain energy. Many metabolic pathways are undisclosed and many organism-specific enzymes involved in metabolism are misplaced. When predicting a specific metabolic pathway of a microorganism, the first and foremost steps is to explore available online databases. Among many online databases, KEGG and MetaCyc pathway databases were used to deduce trehalose metabolic network for bacteria Variovorax sp. PAMC28711. Trehalose, a disaccharide, is used by the microorganism as an alternative carbon source.ResultsWhile using KEGG and MetaCyc databases, we found that the KEGG pathway database had one missing enzyme (maltooligosyl-trehalose synthase, EC 5.4.99.15). The MetaCyc pathway database also had some enzymes. However, when we used RAST to annotate the entire genome of Variovorax sp. PAMC28711, we found that all enzymes that were missing in KEGG and MetaCyc databases were involved in the trehalose metabolic pathway.ConclusionsFindings of this study shed light on bioinformatics tools and raise awareness among researchers about the importance of conducting detailed investigation before proceeding with any further work. While such comparison for databases such as KEGG and MetaCyc has been done before, it has never been done with a specific microbial pathway. Such studies are useful for future improvement of bioinformatics tools to reduce limitations.

Enzymatic Preparation of Non‐Reducing Oligosaccharides from Maltodextrins and Nigerooligosaccharides

AbstractNon‐reducing oligosaccharides with α‐1,1‐ linkages possess unique physicochemical properties, including emulsion stabilizing, moisturizing, pH, and thermal stability. Owing to their non‐reducing quality, they generate fewer colors and flavors when heated through participation in the Maillard reaction. In this study, an enzymatic preparation of non‐reducing maltooligosyl trehalose from maltodextrins, and 3‐O‐α‐d‐glucosyl trehalose from nigerooligosaccharides (NOS) is developed using maltooligosyltrehalose synthase (MTSase). The non‐reducing oligosaccharides are analyzed using high performance liquid chromatography (HPLC) and the decrease in reducing sugar is determined by 3, 5‐dinitrosalicylic acid (DNS) method. The results showed that maltooligosyl trehalose is prepared from commercial maltodextrin, and the loading of 500 U g–1 maltodextrin of MTSase helps ensure that a large proportion of maltotriose is transformed into the glucosyl trehalose. In addition, the reducing power dropped between 61–78%, which indicates that MTSase has a high application potential in the preparation of maltooligosyl trehalose. To the best of the authors' knowledge, this is the first report using MTSase to directly prepare maltooligosyl trehalose from commercial maltodextrin. However, 3‐O‐α‐d‐glucosyl trehalose is barely detected through HPLC. Therefore, the approach to obtain maltooligosyl trehalose from maltodextrins is achieved, while the attempt to obtain 3‐O‐α‐d‐glucosyl trehalose from NOS is not successful.

Efficient biotransformation and synergetic mechanism of dual-enzyme cascade reaction in nonreducing maltoheptaose synthesis

Although maltodextrin is an important bulk ingredient in global food industries, its use is affected by its intrinsic disadvantages, including uneven polymerization and high reducibility. Herein, a cascade enzymatic process for preparing nonreducing maltoheptaose (N-G7) from β-cyclodextrin (β-CD) was developed using cyclodextrinase (CDase) and maltooligosyltrehalose synthase (MTSase). Both enzymes showed good synergistic catalytic activity, and the applicability was demonstrated in a one-pot reaction. A hypothetical synergetic mechanism of CDase and MTSase through the analysis of enzyme reaction pathways was proposed. There exists three successive speed-limiting steps in the synthesis of N-G7, namely: opening of β-CD, multi-site hydrolysis of N-G7 by CDase, and hydrolysis of α-1,1 glycosidic linkage by MTSase. Amongst these, the third speed-limiting step is the most ineffective, and the second speed-limiting step affects the accumulation of N-G7. Fortunately, the MTSase transglycosidic reaction is dominant during the hydrolysis with CDase and is the key to obtain a high yield of N-G7.

Engineering cyanobacteria as cell factories for direct trehalose production from CO2

Trehalose is a non-reducing disaccharide with a wide range of applications in food, cosmetic, and pharmaceutical industries. Cyanobacteria are promising cell factories to produce biochemicals by using solar energy and CO2. Trehalose is biosynthesized at low intracellular concentrations as a salt-inducible compatible solute in some cyanobacteria. In the current study, we demonstrated the efficient trehalose production without salt induction in cyanobacteria by metabolic engineering. The trehalose transporter 1 (TRET1) from an anhydrobiotic insect (Polypedilum vanderplanki) was successfully expressed in the engineered strains and the intracellular trehalose was efficiently secreted to the medium. As the results, the engineered strain co-expressing maltooligosyl trehalose synthase (MTS), maltooligosyl trehalose trehalohydrolase (MTH) and TRET1 secreted 97% of trehalose to the medium, and the titer was up to 2.7 g/L in 15 days. In addition, 5.7 g/L trehalose was produced by semi-continuous cultivation in 34 days. Taken together, this work demonstrates cyanobacteria can be applied as cell factories for direct sunlight-driven conversion of CO2 into excreted trehalose.

Enhanced the catalytic efficiency and thermostability of maltooligosyltrehalose synthase from Arthrobacter ramosus by directed evolution

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in the preparation of trehalose. Increasing interest in trehalose requires further improvement in the catalytic performance of MTSase in order to increase trehalose yield, but so far, the reports about modification of MTSase revealed that the trahalose yield was increased only by 1%. In this study, the mutant S44 P of MTSase from Arthrobacter ramosus was obtained by directed evolution and showed a decrease in Km to 3.5 mM from 6.6 mM of wild-type, and an increase in kcat/Km by 2.2-fold. Thus, S44 P exhibited a trehalose yield of 76.9 %, which was 6.6 % higher than that obtained by wild-type enzyme. This improved trehalose yield is potentially of great importance for the industrial application. Meanwhile, S44 P exhibits increased thermostability compared to wild-type. We solved the structures of both wild-type and mutant enzymes and evaluated differences in their structures to elucidate the changed properties, which provides the insight for future modification of MTSase.

Improved Activity of Sulfolobus acidocaldarius Maltooligosyltrehalose Synthase through Directed Evolution.

Maltooligosyltrehalose synthase (MTSase) is a key enzyme for the production of trehalose from starch. Thermophilic MTSases offer advantages for trehalose production but suffer from low yield. In this study, directed evolution was used to increase the production of Sulfolobus acidocaldarius MTSase (SaMTSase) in Escherichia coli. Mutant libraries constructed using error-prone polymerase chain reaction were assessed using high-throughput activity assays. Three mutants with enhanced activities were obtained, the best of which (mutant D-4) exhibited 2.4 times greater activity than wild-type SaMTSase. The specific activity and catalytic efficiency of D-4 were also greater than those of wild-type SaMTSase. The D-4 activity (624.7 U·mL-1) produced in a 3 L fermenter was 2.0 times greater than that of wild-type SaMTSase. Because the same trehalose yield was obtained using an equal amount of either D-4 or wild-type SaMTSase activity, using D-4 will significantly lower the cost of trehalose production. The activities of the individual mutations present in the three SaMTSase mutants obtained using directed evolution were analyzed. Mutants F284V and T439A exhibited the greatest increases in enzyme activity. Homology models suggested that the decreased side-chain size, weakened hydrophobicity, and decreased interaction might enhance the flexibility of the loop containing catalytic residue Asp443, which was conducive to catalysis.

SpyCatcher/SpyTag-Mediated Self-Assembly of a Supramolecular Complex for Improved Biocatalytic Production of Trehalose

Trehalose has been utilized in the food, cosmetic, and pharmaceutical industries due to its ability to stabilize biomolecules. In this study, we developed a strategy of constructing a self-assembling supramolecular multienzyme complex for the production of trehalose using SpyCatcher and SpyTag. Maltooligosyl trehalose synthase (MTS) and maltooligosyl trehalose trehalohydrolase (MTH) were fused to SpyCatcher and its corresponding ligand, SpyTag. The fused MTS-SpyCatcher (MTS-SC) and MTH-SpyTag (MTH-ST) self-assembled into a supramolecular multienzyme complex (MTS-MTH) via the isopeptide bonds formed between SpyCatcher and SpyTag. MTS-MTH showed the faster reaction rate, which reached the peak of trehalose conversion rate 10 h earlier than the free enzymes. The results demonstrate that the multienzyme self-assembly via the protein-peptide interactions of SpyCatcher and SpyTag can increase the catalytic efficiency of sequential reactions.

Improved Thermostability of Maltooligosyltrehalose Synthase from Arthrobacter ramosus by Directed Evolution and Site-Directed Mutagenesis.

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production. MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use. In this study, mutant G415P was obtained by directed evolution and S361R/S444E was subsequently generated based on a structure analysis of the region around G415. The t1/2 of G415P and S361R/S444E at 60 °C increased by 3.0- and 3.2-fold, respectively, compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was obtained through a combination of the above mutants, and its t1/2 significantly increased by 19.7-fold. Kinetic and thermodynamic stability results showed that the T50 and Tm values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with those of the wild-type enzyme. When the triple mutant was used in trehalose production, the yield reached 71.6%, higher than the 70.3% achieved with the wild-type. Thus, the mutant has a potential application for industrial trehalose production.

Biotechnical production of trehalose through the trehalose synthase pathway: current status and future prospects.

Trehalose (α-D-glucopyranosyl-(1 → 1)-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond. It possesses physicochemical properties, which account for its biological roles in a variety of prokaryotic and eukaryotic organisms and invertebrates. Intensive studies of trehalose gradually uncovered its functions, and its applications in foods, cosmetics, and pharmaceuticals have increased every year. Currently, trehalose is industrially produced by the two-enzyme method, which was first developed in 1995 using maltooligosyltrehalose synthase (EC 5.4.99.15) and subsequently using maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), with starch as the substrate. This biotechnical method has lowered the price of trehalose and expanded its applications. However, when trehalose synthase (EC 5.4.99.16) was later discovered, this method for trehalose production using maltose as the substrate soon became a popular topic because of its simplicity and potential in industrial production. Since then, many trehalose synthases have been studied. This review summarizes the sources and characteristics of reported trehalose synthases, and the most recent advances on structural analysis of trehalose synthase, catalytic mechanism, molecular modification, and usage in industrial production processes.

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

The α-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp. and used as a potent drug in the treatment of type-2 diabetes. In order to improve the yield of acarbose, an efficient genetic manipulation system for Actinoplanes sp. was established. The conjugation system between E. coli carrying ØC31-derived integrative plasmids and the mycelia of Actinoplanes sp. SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture (mycelia and E. coli), quantity of recipient cells, donor-to-recipient ratio and the concentration of MgCl2, which resulted in a high conjugation efficiency of 29.4%. Using this integrative system, a cloned acarbose biosynthetic gene cluster was introduced into SE50/110, resulting in a 35% increase of acarbose titer from 2.35 to 3.18 g/L. Alternatively, a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm) was constructed for gene inactivation, which has a conjugation frequency as high as 0.52%. Meanwhile, almost all 5-flucytosine-resistant colonies were sensitive to apramycin, among which 75% harbored the successful deletion of targeted genes. Using this replicating vector, the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated, and component C was eliminated as detected by LC-MS. Based on an efficient genetic manipulation system, improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

A comparative study of maltooligosyltrehalose synthase from Sulfolobus acidocaldarius expressed in Pichia pastoris and Escherichia coli

Abstract Two genes encoding the maltooligosyltrehalose synthase (MTSase) from Sulfolobus acidocaldarius ATCC 33909 were synthesized: one (designated E-treY) using codons preferred by Escherichia coli and the other (designated P-treY) using codons preferred by Pichia pastoris. E-treY was inserted into pET-24a(+), and the resulting plasmid was used to transform E. coli BL21(DE3). P-treY was inserted into pPIC3.5 K and pGAPZA, and the resulting plasmids were used to transform P. pastoris KM71. The maximum MTSase activity expressed by these E. coli and P. pastoris strains reached 31.6 and 133.0 U mL−1, respectively. The purified MTSases displayed the same pH (pH 5.5) and temperature (75 °C) optima. Both had a half-life of 12 h at 75 °C. The recombinant P. pastoris KM71/pPIC3.5K-treY strain producing the highest enzyme activity was used to optimize the fermentation conditions in a 3.6-L bioreactor. Fermentation for 96 h under optimal conditions yielded an MTSase activity of 747.4 U mL−1.

Genome Analysis of a New Rhodothermaceae Strain Isolated from a Hot Spring

A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50–60°C and is capable of growing in marine broth containing 1–10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (<90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity (ANI) values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na+/H+ antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K+ channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium.

Over-expression of BvMTSH, a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase, enhances drought tolerance in transgenic rice

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]

Identification of substrate-binding and selectivity-related residues of maltooligosyltrehalose synthase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in the synthesis of trehalose. Computer simulations using AutoDock and NAMD were employed to assess the substrate-binding and selectivity-related residues of MTSase. We introduced mutations at residues D411, D610, and R614 to determine the substrate-binding residues of Sulfolobus solfataricus ATCC 35092 MTSase, and introduced mutations at residues P402, A406, and V426 to investigate the enzyme's selectivity-related residues. Kinetic studies of D411A, D610A, and R614A MTSases reveal significant reductions in catalytic efficiency and cause increase in the transition-state energy of mutant MTSases, indicating that residues D411, D610, and R614 form hydrogen bonds to the substrate. Compared with wild-type MTSase, the hydrolysis: transglycosylation selectivity ratio was significantly decreased for P402Q and significantly increased for A406S MTSases, while the ratio for V426T MTSase showed little change. The results suggest that P402 and A406 residues are selectivity-related.