The noncatalytic triad of alpha-amylases: a novel structural motif involved in conformational stability

Chloride-activated alpha-amylases contain a noncatalytic triad, independent of the glycosidic active site, perfectly mimicking the catalytic triad of serine-proteases and of other active serine hydrolytic enzymes. Mutagenesis of Glu, His, and Ser residues in various alpha-amylases shows that this pattern is a structural determinant of the enzyme conformation that cannot be altered without losing the intrinsic stability of the protein. (1)H-(15)N NMR spectra of a bacterial alpha-amylase reveal proton signals that are identical with the NMR signature of catalytic triads and especially a deshielded proton involving a protonated histidine and displaying properties similar to that of a low barrier hydrogen bond. It is proposed that the H-bond between His and Glu of the noncatalytic triad is an unusually strong interaction, responsible for the observed NMR signal and for the weak stability of the triad mutants. Furthermore, a stringent template-based search of the Protein Data Bank demonstrated that this motif is not restricted to alpha-amylases, but is also found in 80 structures from 33 different proteins, amongst which SH2 domain-containing proteins are the best representatives.     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

L-O-Methylthreonine-Resistant Mutant of Arabidopsis Defective in Isoleucine Feedback Regulation

Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M2 populations of ethyl methane sulfonate-treated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to L-O-methylthreonine, an isoleucine structural analog. Growth in the mutants was 50- to 600-fold more resistant to L-O-methylthreonine than in the wild type. The resistance was due to a single, dominant nuclear gene that was denoted omr1 and was mapped to chromosome 3 in GM11b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, Km, Vmax, and pH optimum) of TD in extracts from the wild type and GM11b were similar except for the inhibition constant of isoleucine, which was 50-fold higher in GM11b than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from GM11b than in extracts from wild type. Therefore, isoleucine feedback insensitivity in GM11b is due to a mutant form of the TD enzyme encoded by omr1. The mutant allele omr1 of the line GM11b could provide a new selectable marker for plant genetic transformation.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.     

Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates

Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.     

Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.     

Novel insights on the mechanism of action of alpha-amylase inhibitors from the plant defensin family

Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.     

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.     

Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus beta-glucosidase: effects of donor concentration, water content, and temperature on activity and selectivity in hexanol.

The transglucosylation reaction catalyzed by wild-type beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-beta-glucoside to hexyl-beta-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The selectivity towards transglucosylation was quantified by the S value defined as follows: S = r(S) x a(W)/r(H) x a(hex) where r(S) and r(H) are the initial rates of transglucosylation and hydrolysis and a(w) and a(hex) are the thermodynamic activities of water and hexanol. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-beta-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95 degrees C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-beta-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (a(w)), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as a(w) = 0.29. Surprisingly, the S value increased with increasing water activity up to a(w) = 0.92. At still higher water content the S value decreased.     

N‐β‐alanyldopamine metabolism,locomotor activity and sleep in Drosophila melanogaster ebony and tan mutants

Drosophila melanogaster Meigen mutants for N‐β‐alanyldopamine (NBAD) metabolism have altered levels of NBAD, dopamine and other neurotransmitters. The ebony¹ mutant strain has very low levels of NBAD and higher levels of dopamine, whereas the opposite situation is observed in the tan¹ mutant. Dopamine is implicated in the control of movement, memory and arousal, as well as in the regulation of sleep and wakefulness in D. melanogaster. N‐β‐alanyldopamine, which is best known as a cuticle cross‐linking agent, is also present in nervous tissue and has been proposed to promote locomotor activity in this fly. The daily locomotor activity and the sleep patterns of ebony¹ and tan¹ mutants are analyzed, and are compared with wild‐type flies. The tan¹ mutant shows reduced locomotor activity, whereas ebony¹ shows higher levels of activity than wild‐type flies, suggesting that NBAD does not promote locomotor activity. Both mutants spend less time asleep than wild‐type flies during night‐time; ebony shows more consolidated activity during night‐time and increased sleep latency, whereas tan is unable to consolidate locomotor activity and sleep in either phase of the day. The daily level of NBAD‐synthase activity is measured in vitro using wild‐type and tan¹ protein extracts, and the lowest NBAD synthesis is observed at the time of higher locomotor activity. The abnormalities in several parameters of the waking/sleep cycle indicate some dysfunction in the processes that regulates these behaviours in both mutants.     

定向进化改造酪氨酸解氨酶提高大肠杆菌合成对香豆酸产量

对香豆酸是一种具有多种药理活性的天然酚类化合物,也是多种天然药用产物生物合成的前体物质,广泛应用于食品、化妆品、医药等领域。通过微生物合成对香豆酸相对于化学合成和植物提取工艺具有节能减排等优势。但是,目前微生物合成对香豆酸产量较低,难以满足大规模工业发酵生产的要求。为了进一步提高对香豆酸产量,对粘红酵母酪氨酸解氨酶 (Tyrosine ammonia-lyase,TAL) 进行定向进化改造,利用高通量筛选方法从随机突变体文库中筛选TAL催化活性提高的突变体。通过初筛和复筛两轮筛选,从大约10 000个突变体中获得1个TAL催化活性提高1倍的突变体。该突变体包含3个氨基酸突变位点,分别为S9Y、A11N、E518A。进一步通过单点氨基酸饱和突变验证,当S9位点突变为Y、I、N和A11位点突变为N、T、Y时,TAL的催化活性提高1倍以上。通过对S9和A11位点3种类型突变进行组合突变验证,S9Y/A11N和S9N/A11Y突变体的TAL催化活力显著高于其他组合。将S9N/A11Y突变体质粒转入酪氨酸高产菌株CP032。通过摇瓶发酵,该菌株在48 h时的对香豆酸产量达到394.2 mg/L,比对照菌提高2.2倍。本研究工作对促进微生物合成对香豆酸的代谢工程研究具有一定的参考价值。     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

A dominant-negative Galpha mutant that traps a stable rhodopsin-Galpha-GTP-betagamma complex

Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.     

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Synthesis and biological activity profile of new analogues of the cyclic opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO2)-D-Cys]-NH2 containing (S)-alpha-hydroxymethylcysteine in place of cysteine.

To examine the effect on biological activity of replacing D-Cys in the opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]-NH(2) in position 2 or/and 5 with alpha-hydroxymethylcysteine (alpha-Hmc), three analogues were synthesized. These compounds exhibit agonist activity at both mu and delta receptors. However, the most active analogue, with (S)-alpha-Hmc residue in position 5, was 3360- and 2190-fold less active than the parent peptide in the GPI and MVD assays, respectively.     

Directed evolution of cutinase using in vitro compartmentalization

High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2?C95 and 0?C5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0?C5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1

Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.