臺灣博碩士論文加值系統

由Agrobacterium radiobacter這株細菌所產生的N-carbamoyl-D-amino acid amidohydrolase酵素可應用於工業中以生產D型胺基酸，此酵素目前已有一1.95 Å 的晶體結構，其中一參與酵素催化反應的胺基酸殘基Cys172，被推測可能參與親核性的攻擊，攻擊受質carbamoyl group上的碳原子，借由對受質分子與此酵素的立體結構的分子模擬，我們可大致了解此酵素與受質可能的結合位置及其與胺基酸殘基Cys172的關係，進而透過對Cys172的定點突變所得到的兩個突變酵素，分別為Cys172Ala以及Cys172Ser，我們獲得了這兩個突變酵素的晶體結構，並且分別將N-carbamoyl-D-p-hydroxyphenylglycine (D-NCHPG)受質與突變蛋白作用後進行養晶實驗，解得了受質與突變酵素複合體的晶體結構，經由對突變酵素及其與受質結合的複合體晶體結構的分析，我們了解到以下四點：一、突變後的酵素結構或是具有受質結合於其中的結構，與原本的酵素結構在整體結構上並無差異，二、突變殘基的支鏈結構維持與原殘基的支鏈結構相同的方位，三、在複合體結構中發現數個殘基，其中包含了Asn173. Arg175, Arg176, Glu47, Lys127, His144 和 Glu146參與了與受質結合相關的角色，四、酵素的催化機制與Cys172在催化中所扮演的角色可由晶體結構做更進一步的推測。經由對受質結合部位的了解，我們選擇了其中參與結合受質上carboxyl moiety 的蛋白質殘基Asn173. Arg175, Arg176做定點突變，獲得Asn173Ala、Arg175Ala、 Arg176Ala等突變酵素，經由酵素活性分析發現Arg175Ala、 Arg176Ala完全失去活性而Asn173Ala酵素的活性下降，此外我們也獲得了Arg175Ala以及Asn173Ala兩突變的晶體結構，由對R175A與野生種酵素的結構比較，發現在受質結合位置上，殘基Asn173以及Arg175皆改變了其支鏈的方位，而於N173A之晶體結構中，並沒有發現酵素催化區中有較大的結構變化，然而由酵素動力學的分析卻發現N173A酵素的Km值比起野生種酵素約下降了一半，而其kcat值則上升，另外我們也獲得了Arg175Lys以及Arg176Lys的點突變酵素，此兩酵素的Km值皆上升了二到三倍，而其中R175K的kcat值則有很大的上升，由此可知Arg175, Arg176以及Asn173在受質結合以及酵素催化上皆扮演相當重要的角色。

The crystal structure of N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) from Agrobacterium radiobacter, an industrial biocatalyst for the production of valuable D-amino acids, has been determined to a resolution of 1.95 Å. The active site residue Cys172 has been proposed as the residue involved in a nucleophilic attack of the C atom of the carbamoyl group in a substrate. Docking simulation was performed to predict the substrate-binding pocket by using Grid-docking method. Based on the docking model, the Cys172 was substituted with Ser or Ala to inactivate the enzyme but did not abolish the ability to bind the substrate. Both mutant enzymes, Cys172Ser and Cys172Ala have been crystallized and co-crystallized with the substrate, N-carbamoyl-D-p-hydroxyphenylglycine (D-NCHPG). The crystal structures of Cys172Ala and Cys172Ser were determined respectively in its native form and in complex form with N-carbamoyl-D-p-hydroxyphenylglycine. Analysis of these structures suggest that (i) the overall protein folding, side chain conformations and active sites of both mutants, mutant-substrate complexes are isomorphous with those of wild-type enzyme, (ii) the mutated side chains are pointed for catalysis in a similar conformation as that observed for the wild-type D-NCAase, (iii) within the identified substrate binding pockets, a number of residues including Asn173. Arg175, Arg176, Glu47, Lys127, His144 and Glu146 are involved in interacting with the substrate, and (iv) the role of Cys172 in enzymatic activity and the binding mode of the substrate were further confirmed in the crystal structures. Residues Asn173, Arg175, and Arg176 nearby the carboxyl moiety of the substrate were investigated by site-directed mutagenesis. Crystal structures of Arg175Ala and Asn173Ala D-NCAase were obtained. Structural comparisons of wild type D-NCAase and R175A crystal structures reveal significant conformational changes in the active site. No conformational change is found in the active site of N173A crystal structure. Arg175 and Arg176 were further mutated to Lys residues respectively. Kinetics analysis shows no detectable activity of the Arg175Ala and Arg176Ala mutants and increase Km values of the Arg175Lys and Arg176Lys mutants while a decrease in Km value for Asn173Ala mutant. Site-directed mutagenesis results are discussed in light of the crystal structures of mutant D-NCAase and mutant-substrate complexes.

CONTENTS
ABSTRACT 1
中文摘要 2
INTRODUCTION 5
MATERIALS AND METHODS 8
SITE-DIRECTED MUTAGENESIS 8
ENZYME PURIFICATION AND CONCENTRATION 9
CRYSTALLIZATION, DATA COLLECTION AND PROCESSING 11
STRUCTURE DETERMINATION 12
MODEL BUILDING AND REFINEMENT 13
CIRCULAR DICHROISM OF WILD TYPE AND MUTANT D-NCAASE PROTEINS 14
ENZYME KINETICS ASSAY 15
MODELING OF THE D-NCAASE-SUBSTRATE COMPLEX 15
RESULTS 17
THE DOCKING MODEL AND MUTAGENIC TARGETS 17
ENZYMATIC ANALYSIS AND CRYSTALLIZATION 17
CRYSTAL STRUCTURES OF C172A, C172S, R175A AND N173A MUTANT PROTEINS AND PROTEIN WITH BOUND SUBSTRATE 19
CD SPECTROSCOPY OF WILD TYPE AND MUTANT D-NCAASE PROTEINS 21
KINETIC CHARACTERIZATION OF D-NCAASE MUTANTS 22
DISCUSSION 23
THE WILD TYPE, C172S AND C172A D-NCAASE ACTIVE SITES 23
CHARACTERIZATION OF SUBSTRATE BINDING 24
ROLES OF ACTIVE SITE RESIDUES IN CATALYSIS 28
CONCLUSION 30
REFERENCES 31
FIGURES AND TABLES 33

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