Structure and mechanism studies of sugar epimerase from dictyoglomus turgidum

Dissertation for Degree of Doctor Supervisor: Prof Lin-Woo Kang Structure and Mechanism Studies of Sugar Epimerase From Dictyoglomus turgidum Submitted by PHAM TAN VIET August, 2014 Department of Advanced Technology Fusion Graduate School of Konkuk University Structure and Mechanism Studies of Sugar Epimerase fromDictyoglomus turgidum A Dissertationsubmitted to the Department of Advanced Technology Fusion and the Graduate School of Konkuk University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Submitted by PHAM TANVIET April, 2014 This certifies that the Dissertation of PHAM TAN VIET is approved Approved by Examination Committee: Chairman Member Member Member Member May, 2014 Graduate School of Konkuk University TABLE OF CONTENTS List of Tables v List of Figures vi Abstract ix Chapter Structure and mechanism studies of sugar epimerase from Dictyoglomus turgidum 1.1 Introduction 1.1.1 The important roles of carbohydrate in life 1.1.2 Epimerization reaction mechanism 1.1.3 Three-dimensional structure determination methods 10 1.1.3.1 Electron microscopy 10 1.1.3.2 Atomic force microscopy 12 1.1.3.3 Nuclear magnetic resonance (NMR) 13 1.1.3.4 X-ray crystallography 14 1.1.3.4.1 Protein crystallization principle 16 1.1.3.4.2 Vapor diffusion methods for protein crystallization 18 1.2 Methods 20 1.2.1 Cloning of cellobiose 2-epimerase 20 1.2.2 Overexpression and Purification of cellobiose 2-epimerase 23 1.2.3 Crystallization and X-ray data collection 25 1.2.4 Structure determination and refinement 29 i 1.3 Results and Discussions 31 1.3.1 Quality of the refined model 31 1.3.2 Overal structure of DT_epimerase 34 1.3.3 Epi_DT complex structure 40 Chapter Structure determination of Malonyl CoA-acyl carrier protein transacylase (XoMCAT) 55 2.1 Introduction 55 2.2 Methods 56 2.2.1 Crystallization and X-ray data collection 56 2.2.2 Structure determination and refinement 58 2.3 Results and Discussion 58 2.3.1 Overall structure of XoMCAT 58 2.3.2 Active site structure of XoMCAT 62 2.3.3 Proposed catalytic mechanism of XoMCAT 63 2.3.4 Computational protein-protein docking of XoMCAT and ScACP 64 Chapter Structure determination of Cytochrome P450 107W1 (CYP107W1) 68 3.1 Introduction 68 3.2 Methods 72 3.2.1 Crystallization and X-ray data collection 72 2.2.3 Structure determination and refinement 73 3.3 Results and Discussion 74 3.3.1 Quality of the refined model 74 ii 3.3.2 Overall structure of CYP107W1 77 3.3.3 CYP107W1 complex structure 81 References 84 Supplemental crystallization data 103 Abstract (in Korean) 137 Acknowledgment 139 Publications 141 iii List of Tables Table 1.1Data collection statistics of Epi_DT 33 Table 1.2Interactions between lactose and Epi_DT 47 Table 2.1Datacollection statistics of XoMCAT .57 Table 3.1Datacollection statisticsof CYP107W1 75 Table S.1Data collection statistics of CjPDF 105 Table S.2Data collection statistics of AbALR 110 Table S.3Datacollection statistics of ADH 114 Table S.4Datacollection statistics of XometB 118 Table S.5Datacollection statistics of SFC-1 122 Table S.6Data collection statistics of XoGroEL 126 Table S.7Datacollection statistics of XoGroES 131 Table S.8Datacollection statistics of AbDdl 135 iv List of Figures Figure 1.1Major pathways in carbohydrate metabolism Figure 1.2Epimerase mechanism in general Figure1.3Proposed catalytic mechanisms for AGE and YihS Figure1.4Schematic drawing of electron microscopy apparatus 11 Figure 1.5Work flow for solving the protein structure by X-ray crystallography .15 Figure1.6Schematic illustration of a protein crystallization phase diagram .17 Figure1.7Crystalsin the reservoir solution 19 Figure1.8pET-29b-His-Tev vector sequence 22 Figure1.9Purified His-tagged Epi_DT and untagged Epi_DT 24 Figure1.10Crystals of His-tagged and untagged Epi_DT 28 Figure1.11Diffraction images of His-tagged (a) and untagged (b) Epi_DT .30 Figure1.12Ramachandran plot of ϕ and ψ dihedral angles of Epi_DT .32 Figure 1.13 Overall structure of Epi_DT 35 Figure 1.14Structure-based amino acid sequence alignment 36 Figure1.15Superimposedstructures of epimerases 38 Figure1.16Map of temperature factor of epimerases .39 Figure 1.17Surface map of catalytic center in Apo and lactose-Epi_DT structure 41 Figure1.18Overall structure of Epi_DT with its ligand 43 Figure1.19Interactions between ligand and important residues .44 Figure1.20Interaction between Epi_DT β1 strand and non-reducing sugar region 45 v Figure1.21Conserved important residues interact with reducing sugar region in the active sites of Epi_DT and cellobiose 2-epimerase from Rhodothermus marinus .46 Figure1.22Proposed catalytic reactions of Epi_DT 49 Figure1.23Proposed catalytic intermediates by Epi_DT 50 Figure1.24Ring opening process 51 Figure1.25Proposed epimerization mechanism 52 Figure1.26His247, Glu250, and His188 residues involved in the isomerization 53 Figure1.27Key residues in inter-conversion of lactose to epilactose and lactulose 54 Figure2.1Overall structure of XoMCAT 60 Figure2.2Multiple sequence alignment of MCAT structures 61 Figure2.3Protein-protein docking 66 Figure3.1Typical antibiotics having a macrolide ring .69 Figure3.2General aspects of the P450 catalytic cycle .70 Figure3.3Crystals of Apo-CYP107W1 and Oligomycin A-CYP107W1 72 Figure3.4Ramachandran plot of ϕ and ψ dihedral angles of Apo-CYP107W1 74 Figure3.5Overall structure of Apo-CYP107W1 77 Figure3.6Structure-based amino acid sequence alignment 78 Figure3.7Superimposed structures of P450s 79 Figure3.8Superimposedstructures of apo and complexedCYP107W1 81 Figure3.9Interactions between Heme and oligomycin A 82 FigureS.1Initial and optimized single crystals of CjPDF 104 FigureS.2.1Crystal of AbALR with nine different shapes 108 FigureS.2.2AbALR crystals having adequate dimensions 109 vi FigureS.3Initial ADH crystals obtained after three days 113 FigureS.4Crystals of XometB 117 FigureS.5Crystals of SFC-1 121 FigureS.6Optimized XoGroEL crystal 125 FigureS.7Crystals of chaperonine enzyme from X oryzae pv oryzae 130 FigureS.8A single AbDdl crystal 134 vii Supplementaldata Co-chaperonin XoGroES from Xanthomonas oryzae pv oryzae S.7.1 Introduction of XoGroES Bacterial blight (BB) is one of the most serious bacterial diseases found in rice growing tropical countries and results huge production losses all over the world Rice is one of the most important staple foods for human consumption especially in Asian countries In 2006 alone, agriculture reports indicated that in South Korea itself BB results production losses of rice worth more than 100 million dollars Still there is no effective antibacterial drugs have been identified against this disease and it is very essential to find a drug against Xoo to halt rice-production losses The whole genomic sequences of Xoo has been determined [38], and this provides valuable information to select the drug targeted enzymes against BB As the first step of initiating the drug development against Xoo, 95 gene coding essential enzymes have been selected [39, 40] from the 4,538 putative genes [38] for the drug candidates The selected target genes have been cloned and expressed in Escherichia coli systematically to obtain the essential target enzymes to find atomic resolution structure and protein-drug interaction study by X-ray crystallographic method As the 3D structures are important to understand the reaction mechanism and for drug development, GroES (Xoo4289) gene was cloned and expressed in Escherichia coli Protein misfolding in living cells may lead to malfunctioning of the cell machinery Not only they fail to perform their biological function, but also tend to interact with other biomolecules and disrupt the normal activity of the cell To avoid 128 this misfolding of proteins, naturally both prokaryotic and eukaryotic cells have developed ‘‘chaperone’’ protein complexes that capture misfolded proteins and refold to active proteins, thereby preventing them from indulging in cellular mischief [129-131] Chaperonins are ubiquitous in nature that control the protein folding denatured by various stresses, such as heat shock [129] One of the most important chaperone complexes is the GroEL-GroES complex It consists of two types of proteins, GroEL (~60 kDa) and GroES (~10 kDa) The GroEL has the architecture of tetradecameric oligomer, which consists of two cylindrical chambers, cis and trans rings joined together in symmetrical fashion at the bottom GroES forms a dome-shaped heptamer Sigler and coworkers [140] proposed the action of this chaperone complex; initially, the “open barrel” GroEL hydrophobic inner surface will capture the target misfolded proteins In the next step, ATP-dependent GroEL barrel is capped by heptameric GroES cap The nonnative protein is now trapped inside the hydrophilic folding cavity and the protein is allowed to refold during the time of hydrolysis of ATP to ADP Upon binding of ATP to the trans ring, the GroES cap is released and then expected to release the folded protein The GroEL-GroES complex structure from Escherichia coli is a well learn chaperonin system (PDB ID: 1AON; [141]) Among many Co-chaperonines, GroES structures alone have been reported from Mycobacterium tuberculosis (PDB ID: 1P3H; [142]) and Mycobacterium leprae (PDB ID: 1LEP; [143]) All these GroES structures are homo-heptameric and show dome-shaped architecture, and each monomer consists of small -barrel with a highly flexible mobile loop The mobile loop of GroES from E coli helps to contact the structure of GroEL tetradecamer In 129 addition to this, GroES has some more important functions such as it acts like an immunogen (M tuberculosis and M leprae) [144], it forms tetradecamer with divalent cations (M tuberculosis) [142] and it is more stable in higher temperature up to 80C (Thermus thermophiles) [145] S.7.2 Crystallization and X-ray data collection High throughput crystallization screening of Co-chaperonine (XoGroES) protein produced well diffracting crystal with the condition of 16 % (v/v) PEG 400, 0.2M sodium citrate pH 4.1 and 0.2 M Li2SO4; % (v/v) acetone A complete set of data was collected to 2.0 Å resolution from a single crystal The data analysis and systematic absences suggest that the crystal belongs to hexagonal space group P61 The crystal volume of XoGroES’s asymmetric unit is compatible with single monomer molecule in the unit-cell, with a volume per unit molecular weight of the protein of 2.05 Å Da-1 and a calculated solvent content of 39.9 % (Matthews, 1968) In order to confirm the crystal symmetry, the self-rotation functions were calculated at  = 60, 90, 120 and 180 to detect six-fold, four-fold, three-fold and two-fold axes According to self-rotation function, XoGroES crystal symmetry has been proved to possess two-fold and six-fold symmetries (Because the spacegroup is P61, it should have three-fold axe as well as 6-fold and 2-fold) A preliminary structure solution of the XoGroES protein was obtained by molecular – replacement (MR) using MOLREP program with the crystal structure of chaperonine protein (PDB code 1WNR) from Thermus thermophilus as a model structure The best MR model was derived, which gives a correlation co-efficient of 56.2% and an R factor of 47.4 % at 15 - 3.5 Å resolutions The analysis of best MR solution model fitting 130 with electron density showed good crystal packing and no clashes were found between symmetry-related molecules The resulted solution model was further improved by rigid body refinement Then the structure was mutated with the original amino acid residues of XoGroES and further model is currently being refined by restraint refinement Our final refined structure of XoGroES will provide an insight into its enzymatic reaction mechanism and its role in the chaperonine activity and this information may help us to develop the antibacterial drugs against Xoo Figure S.7 Crystals of chaperonine enzyme from X oryzae pv oryzae (Xoo4289) obtained by sitting drop vapor-diffusion setup using the conditions, Wizard-I, condition 39, 20% (w/v) PEG 1000, Phosphate citrate pH 4.2, 0.2M Li2SO4, The scale bars represents 0.15 mm Co-chaperonine (XoGroES) crystal chosen for crystallographic study from X oryzae pv oryzae (Xoo4289) yielded from16% (v/v) PEG 400, 0.2M Sodium citrate pH 4.1, 0.2M Li2SO4, 4% (v/v) acetone (additive) The scale bars represents 0.1 mm Table S.7 Data collection statistics of XoGroES 131 Synchrotron PAL 6C1 Wavelength (Å ) 0.96418 Resolution Range (Å ) 55.8 - 2.0 Space group P61 Unit-cell parameters (Å ) a&b 64.4 c 36.5 Total No of reflections 51450 No of unique reflections 6225 Completeness (%) 100.0 (100.0) Molecules per AU VM (Å Da-1) 2.05 Solvent content (%) 39.9 Average I/(I) 12.3 (2.8) Rmerge† (%) 8.4 (63.0) †Rsym = hkliIi(hkl) – /hkliIi(hkl), where I(hkl) is the intensity of reflection hkl, hkl is the sum over all reflections, and i is the sum over i measurements of reflection hkl Values in parentheses are for highest-resolution shell 132 Supplementaldata D-alanine-D-alanine ligase from OXA23-producing Acinetobacter baumannii K0420859 S.8.1 Introduction of D-alanine-D-alanine ligase Acinetobacter baumannii is a Gram-negative short, round or rod shaped bacteria A baumannii causes bacteremia, pneumonia, and other respiratory and urinary tract infections in humans and has become an important causative agent of nosocomial infections The ability of A baumannii to improve its resistance mechanisms and survival time makes it difficult to eradicate A baumannii infections from the clinical system [146, 147] Carbapenems have been widely used to treat A baumannii infections, but a worldwide trend of increasing resistance to these antibiotics associated with the production of acquired carbapenemhydrolyzing OXA-type class D beta-lactamases has been reported Until recently, A baumannii strains resistant to carbapenem have been identified and grouped in to four main groups as per the beta lactamase produced by them: OXA-23, OXA-24, OXA-51, and OXA-58 [148] I analyzed OXA-23-producing A baumannii K0420859 (A baumannii OXA23) strain carrying the bla OXA-23 gene primarily located on plasmids or intergrons [149] A baumannii OXA-23 strain was first identified in Spain in 2010 [147] In the case of other carbapenem-resistant A baumannii strains, no drugs that can completely inhibit A baumannii OXA-23 have been found D-Alanine-D-Alanine ligase catalyzes the formation of the precursor of peptidoglycan, an essential component of the bacterial cell wall, which has been a novel target for antibacterial 133 drug development [150] Recently, several antibiotics directed at inhibiting bacterial cell-wall synthesis have been developed Here I performed the cloning of D-Alanine-D-Alanine ligase (AbDdl) gene of A baumannii OXA-23, and the expression, purification, crystallization of AbDdl, and the preliminary X-ray crystallographic analysis of its crystals The atomicresolution structure of AbDdl will be helpful in developing a novel antibacterial drug against A baumannii OXA-23 S.8.2 Crystallization and X-ray data collection AbDdl was successfully overexpressed and purified by performing affinity chromatography and anion-exchange chromatography The homogeneity of the purified protein was examined by performing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) Only one band was visible on the SDS-PAGE after two-step purification, indicating that the purified protein has a molecular weight of about 35 kDa, which is in agreement with the predicted molecular weight of 34.4 kDa X-ray diffraction data were collected up to a resolution of 2.8 Å The crystallographic orthorhombic space group of P212121 with the cell parameters of a = 113.4 Å , b = 116.7 Å , c = 176.5 Å was determined by auto-indexing [151] According to the Matthews coefficient calculation, there are six molecules in the asymmetric unit with a solvent content of 56.3% [152] Analysis of the self-rotation peaks revealed the presence of three two-fold, two three-fold, and one four-fold rotation axes, in addition to three crystallographic twofold axes (Fig S.8) Crystal structure was determined via molecular replacement (MR) by using the AutoMR of the PHENIX package of a crystallography program 134 [86, 153] The search model was generated using the Chainsaw in CCP4 package [154, 155] and D-alanine-D-alanine ligase from Yersinia pestis (PDB ID: 3V4Z; 53% sequence identity) The MR was successful, and it indicated the presence of six protomers in the asymmetric unit The MR solution model was inputted in to AutoBuild [86, 156-160] from the PHENIX package The non-crystallographic rotation axes were confirmed with the MR solution model (Table S.8) The current model with R-factor of 26.0% and R-free of 30.0% showed good crystal packing The AbDdl structure thus determined will be useful in developing an antibacterial drug against A baumannii Figure S.8 A single AbDdl crystal 135 Table S.8 Data collection statistics of AbDdl Synchrotron PAL 7A Wavelength (Å ) 0.97935 Detector ADSC Q270 CCD Temperature of data collection (K) 100 Crystal-to-detector distance (mm) 350 Rotation range per image (°) Total rotation range (°) 360 Exposure time per image (s) Resolution range (Å ) 50.0–2.8 (2.85–2.80) Space group P212121 Unit-cell parameter (Å ) a=113.4; b=116.7; c=176.5 Total no of reflections 413,163 No of unique reflections 55,982 Completeness (%) 96 (92) Molecules per asymmetric unit VM (Å Da-1) 2.8 Solvent content (%) 56.3 Average I/σ (%) 12.9 (1.6) Rmerge (%)† 9.1 (42.0) Multiplicity 7.4 (3.7) †Rsym = hkliIi(hkl) – /hkliIi(hkl), where I(hkl) is the intensity of reflection hkl, hkl is the sum over all reflections, and i is the sum over i measurements of reflection hkl Values in parentheses are for highest-resolution shell 136 Abstract (in Korean) 딕티오글로머스터기둠의당에피머라아제의구조및메 커니즘연구 Pham, Tan Viet 신기술융합학과 건국대학교대학원 딕티오글로머스터기움의당에피머라아제는셀바이오스를 4-O-β-D-글 루코피라노실-D-매노우즈와 4-O-β-D-글루코피라노실-D-푸룩토즈로전환하며, 락토우즈를에피락토우즈와락툭로우즈로전환한다 이효소의아포구조및기질 과복합체구조가결정되었다 구조는 1.8 Å 과 2.0 Å 의레졸루션으로결정되었 고컴플랙스구조에서는환원부위의구조인글루코즈가열린상태로 3개의히스티 딘잔기 (188, 247 및 377)을통해서결합하고있었다 구조의분석결과로 H377과 Y114 잔기가기질의링오픈에관여할것으로예상되며, H247, H377 및 H188의잔 기가에피머라아제활성에작용할것으로추측되어진다 마지막단계인아이소머 라아제반응은 H247, H188 및 E250을통해이루어질것으로구조적으로설명되어 진다 이연구결과는딕티오글로머스터기움의당에피머라아제를산업적으로이 137 용하는데매우유용하게쓰여질것이다.벼흰잎마름병균으로부터말로닐코앤자 임에이-아실캐리어단백질트랜스아실라아제 (XoMCAT) 효소의구조가 2.3 Å 의레졸루션으로 N-사이클로핵실-2-아미노에탄설포네이트와복합체구조로결 정되었다 MCAT은말로닐코앤자임에이를아실캐리어단백질에전달하는역할 을하며, 그활성은항생제개발에중요한타깃이다 스트렙토마이시스에버미티 스의 CYP107W1은마크로라이드올리고마이신 C를올리고마이신 A로바꾸는 활성을가진다 CYP107W1의삼차원구조가아포및복합체의구조로결정되었다 아포구조는 2.3 Å 의레졸루션으로, 올리고마이신 A와결합된복합체구조는 2.3 Å 의레졸루션으로결정되었다 중요단어들:당전환효소, 셀바이오스 2-에피머라아제, 말로닐코앤자임에이아실캐리어단백질트랜스아실라아제, 사이토크롬 P450,단백질결정학 138 Acknowledgment With the following lines I wish to express all my gratitude to people who contributed this achievement This dissertation would not have been possible without the support and encouragement received from various quarters First and foremost, I would like to express my deepest gratitude to my supervisor and mentor, Professor Lin-Woo Kang for his invaluable guidance, insightful advices, strong encouragement and generous support for both Ph.D study and my life in Korea I would like to thank my thesis committee members (Prof Ye-Sun Han, Prof Dong-Hak Kim, Dr Sun-Shin Cha and Dr Yeon-Gil Kim) for their time reading my thesis and give useful feedbacks and comments I would like to thank Prof Roger Kornberg and Prof Mario Amzel for their suggestions and discussions I would like to thank all the members in my lab, KU Global lab: Thanh, Thuy, Myoug-Ki, Seung-Hwan, Jun-Ho, Huyen, Duc, Hoang, Hung, and Quynh Especially, I am greatly indebted to Dr Jin-Kwang Kim for his helps and discussions I would also like to thanks my friends in Konkuk University (A Dai, Tri, Truong, Dat, Tuyen, Tien, Hieu, Thong, San, Van Anh, Quang, Thach, Cuong, Tran…) for their support and great friendship I also would like to thank my wife – Dieu Hanh – for all the things that she has shared with me Finally, I would like to thank my parents, sisters, and brother for all their love, 139 affections, emotion support, prayers and encouragement all along my studies I would like to dedicate this work to my parents Seoul, June 08th, 2014 Pham, Tan Viet 140 Publications Pham, T V., Tran, H T., Ngo, H P., Hong, M K., Kim, J G., Lee, S H., Ahn, Y J & Kang, L W (2014) Acta crystallographica Section F, Structural biology communications 70, 604-607 Huynh, K H., Tran, H T., Pham, T V., Ngo, H P., Cha, S S., Chung, K M., Lee, S H & Kang, L W (2014) Acta crystallographica Section F, Structural biology communications 70, 505-508 Pham, T V., Hong, S H., Hong, M K., Ngo, H P., Oh, D K & Kang, L W (2013) Acta crystallographica Section F, Structural biology and crystallization communications 69, 1163-1166 Tran, H T., Pham, T V., Ngo, H P., Hong, M K., Ahn, Y J & Kang, L W (2013) Acta crystallographica Section F, Structural biology and crystallization communications 69, 1120-1122 Ngo, H P., Hong, S H., Hong, M K., Pham, T V., Oh, D K & Kang, L W (2013) Acta crystallographica Section F, Structural biology and crystallization communications 69, 1037-1040 Nguyen, D D., Ngo, H P., Hong, M K., Pham, T V., Lee, J H., Lee, J J., Kwon, D B., Lee, S H & Kang, L W (2013) Acta crystallographica Section F, Structural biology and crystallization communications 69, 1041-1044 Nguyen, H T., Chong, Y., Oh, D K., Heo, Y S., Viet, P T., Kang, L W., Jeon, S J & Kim, D E (2013) Analytical biochemistry 434, 284-286 Kim, S., Nguyen, T D., Lee, J., Hong, M K., Pham, T V., Ahn, Y J., Lee, B M., Han, Y S., Kim, D E., Kim, J G & Kang, L W (2013) Journal of microbiology and biotechnology 23, 22-28 Hong, M K., Lee, J J., Wu, X., Kim, J K., Jeong, B C., Pham, T V., Kim, S H., Lee, S H & Kang, L W (2012) Acta crystallographica 141 Section F, Structural biology and crystallization communications 68, 1124-1127 10 Natarajan, S., Kim, J K., Jung, T K., Doan, T T., Ngo, H P., Hong, M K., Kim, S., Tan, V P., Ahn, S J., Lee, S H., Han, Y., Ahn, Y J & Kang, L W (2012) Molecules and cells 33, 19-25 11 Ngo, H P., Kim, J K., Kim, S H., Pham, T V., Tran, T H., Nguyen, D D., Kim, J G., Chung, S., Ahn, Y J & Kang, L W (2012) Acta crystallographica Section F, Structural biology and crystallization communications 68, 1515-1517 12 Doan, T T., Natarajan, S., Song, N H., Kim, J., Kim, J K., Kim, S H., Viet, P T., Kim, J G., Lee, B M., Ahn, Y J & Kang, L W (2011) Acta crystallographica Section F, Structural biology and crystallization communications 67, 44-47 13 Kim, S H., Lee, S E., Hong, M K., Song, N H., Yoon, B., Viet, P., Ahn, Y J., Lee, B M., Jung, J W., Kim, K P., Han, Y S., Kim, J G & Kang, L W (2011) Journal of microbiology and biotechnology 21, 679-685 142 [...]...Abstract Structure and Mechanism Studies of Sugar Epimerase from Dictyoglomus turgidum Pham, Tan Viet Department of Advanced Technology Fusion Graduate School of Konkuk University Cellobiose 2 -epimerase from Dictyoglomus turgidum( Epi_DT) is an enzyme which inter-convertscellobiose to 4-O-β-D-glucopyranosyl-D-mannose and 4-O-β-Dglucopyranosyl-D-fructose and lactose to epilactose and lactulose.Idetermined... substrate binding pocket Key words: Dictyoglomus turgidum, cellobiose 2 -epimerase, lactose, carbonhydrate.Xanthomonas oryzae pv oryzae,bacterial blight (BB) disease,XoMCAT, acyl carrier protein, Streptomyces avermitilis, CYP107W1, oligomycin ix Chapter 1 Structure and mechanism studies of sugar epimerase from Dictyoglomus turgidum 1.1 Introduction 1.1.1 The important roles of carbohydrate in life Carbohydrates... N-acetyl-D-glucosamine 2 -epimerase (AGE) superfamily In this superfamily, the structures of two epimerases, i.e., AGE and aldose–ketose isomerase YihS have been reported, and their catalytic mechanisms have been suggested [15] The three-dimensional structures of AGEs from Anabaena sp CH1 (PDB ID: 2GZ6)[14], and porcine kidney (PDB ID: 1FP3) [16]have been determined, and the catalytic mechanism of AGE was postulated... reprotonation from the opposite side, and the final reduction of the C3 keto group to give 2 and regenerate the cofactor In mechanism B, the substrate 1 undergoes an anti-elimination of UDP from UDP-GlcNAc to form a 2acetamidoglucal intermediate 2 A subsequent rebound of UDP in a synaddition could form the product UDP-ManNAc 2 Elements from these two mechanisms can be incorporated into a hybrid mechanism (mechanism. .. metabolism of carbohydrates[5] 1.1.2 Epimerization reaction mechanism Many enzymatic transformations of carbohydrates involve the inversion of configuration at one or more stereogenic centers Such an epimerization has been commonly used in the conversion of D-sugars to the corresponding L-sugars It is also a convenient mechanism for accessing the structural diversity derived from a handful of common sugar. .. stabilize the positive charge 8 of their imidazole rings Furthermore, the structure of YihS, which catalyzes isomerization of an unmodified sugar, has been also determined The structures of YihS from Escherichia coli (EcYihS) (PDB ID: 2RGK) and Salmonella enterica (SeYihS) (PDB ID: 2AFA), which show high levels of activity toward mannose and glucose, are similar to that of AGE Although RaCE shows sequence... resolution of determined structure is usually low 13 1.1.3.4 X-ray crystallography X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions From the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of. .. lactulose.Idetermined the Epi_DT structure in apo and complex forms The three-dimensional structure of apo Epi_DT was determinedat 1.8 Å resolution and the complex structure of E250A mutated enzyme with lactose was determined at 2.0 Å resolution, whichshowed an open form of glucose (reducing part) in the catalytic pocket and was bound by 3 important histidine residues (His188, His247, and His377) The structural... oligosaccharide, and enhances proliferation of bifidobacilli and lactobacilli in the gut[10] This stimulation of growth of beneficial bacteria suppresses the conversion of primary bile acid to secondary bile acid, which is considered as a risk factor for colon cancer, and enhances absorption of minerals,including calcium, magnesium, and zinc[11] Furthermore, epilactose increases intestinal absorption of calcium... to AGE and YihS, several amino acids in the catalytic centers of AGE and YihS are well conserved in CE (Arg52, His243,Glu246, Trp249, Trp304, Glu308, and His374 inRɑCE), and mutational experiments with RɑCE indicated that these residues are critical for the catalytic activity[17] However, the substrate specificities of CE, AGE, and YihS differ from each other in terms of substrate chain length and in