애기장대 ureidoglycine aminohydrolase와 ureidoglycolate amidohydrolase의 구조와 기능에 대한 연구

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 8. 이상기.In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine to ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. By using x-ray crystallography, crystal structures of two enzymes in this pathway were solved in this study: (S)-ureidoglycine aminohydrolase (UGlyAH) and the (S)-ureidoglycolate amidohydroalse (UAH), both from Arabidopsis thaliana. UGlyAH enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH) is reported. The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bi-cupin fold of the β-barrel and an octameric functional unit, as well as an Mn2+ ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn2+ ion acts as a molecular anchor to bind (S)-ureidoglycine and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn2+ ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism. In the final step of the pathway, UAH catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, three crystal structures of Arabidopsis thaliana UAH: bound with substrate, reaction intermediate, and product, and a structure of Escherichia coli AAH complexed with allantoate are reported. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-close conformational transition in response to ligand binding, and that the active site size as well as the interaction environments in each UAH and AAH are determinants for substrate specificity between two structurally homologous enzymes.ABSTRACT I CONTENTS III LIST OF FIGURES VI LIST OF TABLES VIII LIST OF ABBREVIATIONS IX CHAPTER I. INTRODUCTION 1 CHAPTER II. Structural and Functional Insights into (S)-Ureidoglycine Aminohydrolase, Key Enzyme of Purine Catabolism in Arabidopsis thaliana 8 MATERIALS AND METHODS 1. Construct 9 2. Protein Expression, Purification and Crystallization 11 3. Data Collection and Structure Determination 14 4. Enzyme Assay 20 5. Metal Analysis 24 RESULTS 1. Molecular Architecture of Octameric AtUGlyAH in a Ligand-Free Form 25 2. Interactions between Monomers in the Intra- and Inter-layers 31 3. Active Site in the Ligand-Free Form of AtUGlyAH 35 4. Binding Mode of (S)-Ureidoglycine in the Binary Complex 42 5. Functional Analysis 48 DISCUSSION 53 CHAPTER III. Structural and Functional Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase from Arabidopsis thaliana and Its Comparison with Allantoate Amidohydrolase 58 MATERIALS AND METHODS 1. Construct 59 2. Protein Expression and Purification 61 3. Crystallization 62 4. Data Collection and Structure Determination 66 5. Enzyme Assay 71 RESULTS 1. Overall Structure of AtUAH 72 2. Active Site with a Bimetal Center 78 3. Binding Mode of (S)-Ureidoglycolate in a Binary Complex with AtUAH(E183A) 81 4. Binding Mode of the Intermediate and Product in the Binary Complex with AtUAH 85 5. Binding Mode of Allantoate in the Closed Conformation of the EcAAH Binary Complex 89 6. Functional Analyses 93 DISCUSSION 96 CHAPTER IV. CONCLUSIONS 103 REFERENCES 105 ABSTRACT IN KOREAN 113 CURRICULUM VITAE 116 PUBLICATIONS 117 ACKNOWLEDGEMENTS IN KOREAN118Docto

한국 정부에서 제도화된 균형성과표(BSC)의 특성 및 그 결정요인에 관한 연구 : 행정자치부의 사례를 중심으로

학위논문(석사)--서울대학교 행정대학원 :행정학과, 행정학전공,2006.Maste

SUH의 활성 변화와 관련한 구조적 변경

Thesis(master`s)--서울대학교 대학원 :농생명공학부,2005.Maste

Effect of the Nuclear Factor I-C on the formation of Hertwig's epithelial root sheath during root development

치아의 형성은 상피-간엽간의 상호작용을 통해 조절되어지는 복잡한 발생과정이다. 지금까지 치관의 발생에 관여하는 유전자 및 그들의 신호전달경로에 관한 연구는 다수 진행되어 왔지만 치근의 발생을 조절하는 기전에 대해서는 별로 알려진 것이 없다. 최근에 NFI-C knock out 생쥐에서 정상치관에 비정상적인 치근을 가지는 치아가 보고되었다. 본 연구의 목적은 NFI-C가 어떻게 치근의 형태와 상아모세포의 분화에 관여하는지를 규명하는 것이다. NFI-C knock out 생쥐의 치근 발생동안에 HERS의 역할을 연구하고자 cytokeratin 면역조직화학적방법과 치근상아질의 특성을 규명하기 위해 DSPP mRNA in-situ hybrydization법을 수행하였다. 1. NFI-C knock out 생쥐의 치근형성시 HERS의 역할 Wild type과 knock out type 모두에서 cytokeratin은 모든 HERS 세포들과 반응하였고, HERS와 법랑상피 사이의 양성반응세포들의 연속성은 치경부 부위에서 소실되었다. Knock out type에서 치근상아질이 침착된 후, cytokeratin 양성-HERS 세포들은 치경부에서 불규칙한 배열과 극성의 상실을 보였다. 2. NFI-C knock out 생쥐의 치근상아질의 특성 DSPP mRNA의 발현은 wild type에서 치관과 치근상아질의 상아모세포 모두에서 강한 발현을 보인 반면, knock out type에서는 치관부위 상아질의 상아모세포에서만 강한 발현을 보였다. 3. NFI-C knock out 생쥐의 치근 발생과정에서 HERS는 치관으로부터 정상적인 확장을 보인 반면, 치근부위에서의 상아 모세포 분화는 실패하였다. 위의 결과들로 보아 NFI-C는 치근형성 과정에서 상아모세포 분화에 중요한 역할을 하는 것으로 사료된다. Tooth formation is a complex developmental process that is mediated through a series of reciprocal epithelial-mesenchymal interactions. Several signal pathways and transcription factors have been implicated in regulating molar crown development, but relatively little is known about the regulation of root development. It was reported that NFI-C knockout mice showed abnormal root formation with normal crown. The aims of this study are to elucidate how the NFI-C regulate the determine of root shape and odontoblasts differentiation. We carried out immunohistochemistry using cytokeratin to investigate the role of Hertwig's epithelial root sheath and DSPP mRNA in-situ hybridization to conform the nature of root dentin during root development in NFI-C knockout mice. Cytokeratin reacted with all the HERS cells and the continuity of cytokeratin positive cells between the HERS cells and enamel epithelium was lost in the cervical region both wild and K/O types. After root dentin deposition cytokeratin positive-HERS cells showed irregularity and loss of polarity in the cervical region in K/O type. DSPP mRNA was strongly expressed in odontoblasts of crown and root dentin in wild type mice, whereas expression of DSPP mRNA was restricted in odontoblast of crown dentin in the K/O type. During root formation in NFI-C knockout mice, HERS normally grow out of the crown but fail to induce odontoblast differentiation in root portion. These results suggest that NFI-C may play important roles in odontoblast differentiation during root dentin formation.위 연구는 조선대학교 연구년제 해외파견(2001년) 지원에 의하여 이루어진 것임

EXPRESSION OF OD314 DURING AMELOBLAST DIFFERENTIATION AND MATURATION

법랑모세포는 법랑질을 형성하고 유지하는 세포로, 법랑질의 유기기질을 분비하고 법랑질 석회화 과정에도 관여한다. 치아 발생과정에서 법랑모세포의 분화는 순차적인 상피-간엽 상호작용에 의하여 조절되나, 분화나 성숙과정의 정확한 기전은 아직까지 잘 알려져 있지 않다. 최근에 상아모세포에서 처음 발견된 OD314가 치아 발생과정에서 상아질을 형성하는 상아모세포 뿐 아니라 법랑모세포에도 발현된다고 하였다. 이에 본 연구에서는 생쥐 하악 전치의 다양한 시기의 법랑모세포를 이용하여, 형태학적 분석과 in-situ hybridization에 의한 OD314 mRNA의 발현 그리고 OD314 항체를 이용한 면역조직화학적 분석을 통하여 OD314유전자의 법랑모세포 분화와 성숙과정에서의 역할을 연구하여 다음과 같은 결과를 얻었다. 1. 형태학적으로 법랑모세포는 분화 단계에 따라 분비 전단계 법랑모세포, 분비기 법랑모세포, 성숙기의 평탄끝 법랑모세포와 성숙기의 주름끝 법랑모세포로 구분되었다. 2. OD314 mRNA는 분비기의 법랑모세포에서부터 발현되기 시작하여 법랑모세포가 성숙해갈 수록 그 발현이 증가하였다. 3. OD314 단백질은 분비 전단계의 법랑모세포에서는 발현되지 않고, 분비기의 법랑모세포에서는 세포질에 전체적으로 발현되었다. 성숙기의 평탄끝 법랑모세포와 주름끝 법랑모세포에서는 세포의 근심과 원심끝단에 OD314 단백질이 강하게 발현되었다. 이상의 결과를 종합하여 OD314는 법랑모세포의 분화와 성숙과정에서 세포질 내부에서 특징적인 역할을 하는 것으로 사료된다. Ameloblasts are responsible for the formation and maintenance of enamel which is an epithelially derived protective covering for teeth. Ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions. However, little is known about the differentiation and maturation mechanisms. OD314 was firstly identified from odontoblasts by subtraction between odontoblast/pulp cells and osteoblast/dental papilla cells, even though OD314 protein was also expressed in ameloblast during tooth formation. In this study, to better understand the biological function of OD314 during amelogenesis, we examined expression of the OD314 mRNA and protein in various stages of ameloblast differentiation using in-situ hybridization and immunohistochemistry. The results were as follows : 1. The ameloblast showed 4 main morphological and functional stages referred to as the presecretory, secretory, smooth-ended, and ruffle-ended. 2. OD314 mRNA was expressed in secretory ameloblast and increased according to the maturation of the cells. 3. OD314 protein was not expressed in presecretory ameloblast but expressed in secretory ameloblast and maturative ameloblast. OD314 protein was distributed in entire cytoplasm of secretory ameloblast. However, OD314 was localized at the proxiamal and distal portion of the cytoplasm of smooth-ended and ruffle-ended ameloblast. These results suggest that OD314 may play important roles in the ameloblast differentiation and maturation.한국과학재단 목적기초연