Structure, Assembly, and Mechanism of a PLP-dependent Dodecameric L-Aspartate β-Decarboxylase

本論文主要是探究Alcaligenes faecalis 菌株中L-天門冬胺酸β-去羧酶(L‐aspartate‐decarboxylase (ASD))之立體結構與酵素功能分析。此類酵素於1950 年起應用於工業酵素造、合成抗生素或食品添加劑中。AsdA 乃一雙功能酵素，屬於磷酸比哆醛第一型(PLP Foldype I)酵素，其主要作用為「去羧基反應」－催化L‐aspartate 形成L‐alanine 及CO2 分子；為「轉胺反應」－以乒乓(ping‐pong)作用方式轉胺於oxaloacetate 上，形成L‐glutamate。究中應用重金屬汞(Hg)之繞射強度差異，以multiple anomalous dispersion(MAD)方式析出AsdA 蛋白結構之相位角(phase angle)，並建構其組成胺基酸之原子空間位置。結果出AsdA 蛋白以雙分子為基本單位，可組裝成12 個分子之truncated tetrahedron 幾何結的巨分子；並具有四個活性中心：受質結合中心(βY‐loop‐βZ)、PLP 催化中心(Lys315)、結調節中心(α13‐α15)及分子聚合中心(α3‐α5及α16‐α17)。藉由沉降係數分析及pH 活性測試，發現具活性之AsdA 蛋白於生物體中以12 分子合結構存在。每單分子分為二個領域(domain)－大領域(L‐domain)及小領域(S‐domain)，大小領域間存在一個磷酸比哆醛(PLP)輔脢分子。於磷酸比哆醛C2 上之甲基與胺基酸ys315 的N4 形成Schiff 鍵，其磷酸根與周圍胺基酸形成氫鍵網，並與α8 雙螺旋結構上的 型環(P‐loop)以偶極之(dipole)力量存在。AsdA 分子結構受到pH 值調控，當pH 小於7 時AsdA 六個雙分子相互緊密結合且活性增加，而pH 增加至8.5 時，使AsdA 解離成無活之雙分子。驗中從PLP結構相對位置設計七個胺基酸突變區(K17A、R37A、Y134F、Y207F、K315A、441A 及R487A)並比較其活性，以探討酵素催化模式。結果發現Y134 提供氫氧基至相鄰體的PLP 催化中心，以氫鍵穩定相鄰單體內的PLP 輔脢分子，喪失此氫氧基團將降低60%羧基的反應效率。除R487A 外，六種突變蛋白平均皆降低30%的反應速率，其中K17AR37A 則提高50%的受質結合力，Y134F 及K315A 則降低40%的受質結合力，Y207F 及Y441F維持60%的催化效率，不影響受質結合力。而Arg487 位於受質結合中心中央，失去正電的側鍊基團(R487A)使AsdA 活性完全喪失，但不影響十二倍體蛋白立體結構的組裝。其i個原子的振動值明顯降低至基本線，表示分子間的移動或振動與原態AsdA 有極大的不，突顯出Arg487 位於重要的樞紐。外，AsdA 酵素活性亦受到pH 值調控，當pH 小於6 時，去羧基反應之活性大幅提50 倍以上。在中性環境下，K17A、R37A 及Y441F 的L‐aspartate 消耗率比原態AsdA 還，與其產物(L‐alanine)含量不成正比，預測L‐aspartate 的消耗原因乃捨去羧基反應且進行胺反應。研究之AsdA 蛋白與Pseudomonase sp.菌株之相似度極高的L-天門冬胺酸β-去羧amp;#37238;(AsdP)具有相似之磷酸比哆醛第一型酵素具有受質引發構型改變(substrate inducedonformational change)現象－晶體加入抑制劑(β‐chloroalanine)浸泡，會引發晶胞(unit cell)小改變，其AsdP 晶格相同，晶胞大小平均單軸增長2.7%，總體積變化高達10%。研究發現此巨分子結構的小領域(S‐domain)構型，因β‐chloroalanine 進入PLP 催化中心，造成 端的胺基酸(1‐38)朝六角形中心(α4 helix bundle)內移，藉凡得瓦力(van der Waals force)引區域性胺基酸(α19、α20 及α21 helices)移動5 Å 距離並轉動約22.5 度。研究論文透過現代科技的x‐ray 繞射技術，並結合結構生物技術及蛋白物理化學分，成功解出完整之AsdA 及AsdP 結構，更進一步探討雙功能酵素作用機制。並預測多功巨分子在生物體中所扮演的角色，包括胺基酸代謝、細胞內重要胺基酸含量之調控及酵功能的轉換，如L‐glutamate 與glutamate decarboxylase 及其反向轉運體(antiporter)之角關係。透過AsdA 及AsdP 酵素的結構的進一步解析，將有助於酵素基礎研究之瞭解，更促進相關科學之發展。The focus of this PhD thesis is the type-I PLP (pyridoxal 5’-phosphate) enzyme-aspartate β-decarboxylase (ASD, from Alcaligenes faecalis) with particular reference ton analysis of protein structure determination and functional activity characterization.he ASD has bi-functional activity. The major one being the conversion of aspartate tolanine and CO2 by decarboxylation, but additionally, it also functions to transaminatespartate to produce oxaloacetate. Similar to the homodimeric aminotransferases, itsrotein subunit comprises a large and a small domain, of 410 and 120 residues,espectively. The crystal structure reveals a dodecamer made of six identical dimershich are arranged in a truncated tetrahedron whose assembly involves tetramer andexamer as intermediates. Based on this structure, we proposed a catalysis mechanismnd four functional motifs: a substrate binding motif (βY-loop-βZ), a PLP binding siteLys315), a regulatory motif (α1- α2 and α13- α15) and an assembly motif (α3- α5 and16- α17). The additional helical motifs I (α3- α5) and II (α16- α17) participate in theligomer formation. Triple mutations of S67R/Y68R/M69R or S67E/Y68E/M69E in motif Iroduced an inactive dimer. The functional dodecamer structure is rather distinct fromhe aminotransferase family. The PLP is bound covalently to Lys315 in the active site,hile its phosphate group interacts with the neighboring Tyr134. Removal of the bulkyide chain of Arg37, which overhangs the PLP group, improved the substrate affinity.utations in flexible regions produced the more active K17A and the completely inactive487A. The structure also suggests that substrate binding triggers conformationalhanges essential for catalyzing the reaction. The substrate induced S-domainonformational change was elucidated by β-chloralanine–AsdP complex. Along thehree-fold axis of ASD structure, there are four 1.4 Å radius in size pores were appearedvn each three α4 helices bundle of the plate shape hexamer. The surface electronotential of α4-α5 helices was changed from most positive charge to half hydrophobicityhat cooperated with N-terminal moved and rotated about 5 Å and 22.5 degree,espectively. The cross-interaction of S-domain involved with van der Waals forceetween α1 to α2, α1 to α20, and α20 to α21 helices those move together byydrophobic patch I, II, and III. The Arg497 residue has observed that was contributedith stabilize carboxyl group of side chain of β-chloralanine-PLP complex as ATase’snzymatic reaction.中文摘要 ........................................................................................................................................ ibstract .......................................................................................................................................... iiibbreviations ................................................................................................................................. vhapter 1 Introduction ................................................................................................................... 1he story of L-aspartate β-decarboxylase ............................................................................. 4mino acid metabolism of ASD ............................................................................................... 5iochemical characterization of ASD ...................................................................................... 7hapter 2 Materials and Methods ............................................................................................. 10ingle and multiple point mutations ...................................................................................... 10rotein expression and purification ....................................................................................... 10ircular dichroism ( CD ) spectroscopy analysis ................................................................ 11olecular weight determination by analytical ultracentrifugation ( AUC ) ....................... 12etermination of the binding number by isothermal calorimeter ( ITC ) ......................... 13rotein particle size determination by dynamic light scattering ( DLS ) .......................... 13V-visible spectrophotometry ................................................................................................ 14etermination of the conformational change by Fluorescence meter ............................. 14etermination of the enzyme kinetics by HPLC .................................................................. 15rystal screening for L-aspartate β-decarboxylase (ASD)………………………………16rystallization conditions, data collection, structure determination and refinement ...... 17 Crystallization conditions .............................................................................................. 17 Data collection ................................................................................................................ 17 Phase strategy and MAD determination .................................................................... 19 Model building ................................................................................................................ 20 Molecular replacement (MR) ........................................................................................ 21 Multiple isomorphism replacement (MIR) .................................................................. 23 Structure refinement ...................................................................................................... 23 PDB accession codes ................................................................................................... 24hapter 3 Results ........................................................................................................................ 25mprovement of X-ray diffraction on AsdA from salt to protein crystal condition ........... 25 Two-step purification for ASD protein ......................................................................... 25 Protein concentration .................................................................................................... 26 Buffer effect ..................................................................................................................... 26 Anti-freeze reagent ........................................................................................................ 27he native reflection data of both ASD ................................................................................. 28olving the ASD structure ....................................................................................................... 29 Using the molecular replacement method ................................................................. 29 Using the isomorphism displacement method .......................................................... 30 Solving the Phase problem by two wavelength MAD method ................................ 31SD structure determination is complicated by two methods .......................................... 32 truncated tetrahedron geometry of the macromolecule ASD ........................................ 33he dimer as a basic unit of dodecamer structure ............................................................. 33he monomer structure comparison ..................................................................................... 34 similar PLP binding site to the AT family ........................................................................... 36he dodecamer structure assembly depends on pH value............................................... 37he important interface structure .......................................................................................... 38ost decarboxylase activity on dimer structure ................................................................... 40he role of PLP surrounding residuals and their activity ................................................... 42ubstrate induced conformational change and mechanism prediction .......................... 45hapter 4 Discussion .................................................................................................................. 50tructure determination ........................................................................................................... 50odecamer enzyme activity ................................................................................................... 51imilar folding with different activity and In vivo function .................................................. 53he bi-functionality of ASD in relation to the substrate interaction face .......................... 55wo base mechanism ............................................................................................................. 58pecialized dodecamer structure .......................................................................................... 59eference ………………………………………………………………………………….……61chemes ....................................................................................................................................... 71cheme 1. The PLP-dependent enzyme catalysis position and torsion angle of internalldimine ................................................................................................................... 72cheme 2. The major stratagem of determining ASD protein structure ............................ 73cheme 3. The proposed mode of dodecamer activity. ....................................................... 74igures .......................................................................................................................................... 75igure 1. The ASD protein expression, purification and crystallization ............................. 76igure 2. The heavy atom site of Hg derivative AsdP structure.......................................... 77igure 3. The buffer effect of oligomer population were elucidated by AUC .................... 78igure 4. The anomalous scattering factor ............................................................................ 79igure 5. The quaternary structure of the dodecamer ......................................................... 80igure 6. Structure alignment of the monomer of AsdA ....................................................... 81igure 7. The amino acid alignment of PLP enzymes ......................................................... 82igure 8. The monomer and dimer structures of ASD ......................................................... 83igure 9. The topology diagram of an AsdA monomer ......................................................... 84igure 10. The PLP binding site of ASD and AT ................................................................... 85igure 11. The active site structure ......................................................................................... 86igure 12 The environment of the active site ........................................................................ 87igure 13. The interface of dimer ............................................................................................ 88igure 14. The pH dependence of the dodecamer assembly of AsdA .............................. 89igure 15 The proposed assembly mechanism of AsdA ..................................................... 90igure 16. The α3-α 5 helices assembly motif ...................................................................... 91igure 17 The pH effect of the three α4 helices bundle and α3-loop-α4 .......................... 92igure 18. The circular dichroism (CD) spectra .................................................................... 93igure 19. The UV/Vis spectra of ASD ................................................................................... 94igure 20. The UV spectra of aldimine PLP reduced by L-aspartate treatment .............. 95igure 21. The B value and RMSD analysis ......................................................................... 96igure 22. The sigmoid kinetic of ASD ................................................................................... 97igure 23 The substrate binding number by ITC determination ......................................... 98igure 24 The comparison of substrate binding site with AT complex .............................. 99igure 25. The flexible N-terminal segment and βY-loop-βz random coil ....................... 100igure 26. The proposed a catalytic mechanism of AsdA ................................................. 101igure 27. The structure of β−chloralanine-AsdP complex ............................................... 102igure 28. The substrate induced S-domain conformational change of ASD complex 103igure 29. The contribution of hydrophobic patch cross-interaction occur in S-domain104igure 30. The ASD metabolism is depend on pH ............................................................. 105ables .......................................................................................................................................... 106able I. The common name of L-Aspartate β−decarboxylase .......................................... 107able II. The ASD protein has been found on different species ....................................... 108able III. Data collection and refinement statistics of the ASD crystals........................... 109able IV. Models of AT and DC as a search model for MR method ................................. 110able V. The list of heavy atom soaking with AsdA crystals .............................................. 111able VI. The slow transformation of unit cell diameter of ASD is depend on soakingime .……………………………………………………………………………… 112able VII. The variant of the three axes distance of AsdA and its mutants ..................... 113able VIII. the list of RMSD comparison of each chains of asymmetry unit ................... 114able IX. Effects of mutations on the kinetic parameters of AsdA .................................... 115able X. Residues at the interface between two dimers in an AsdA dodecamer ........ 116ppendix ..................................................................................................................................... 117ppendix A. The Effects of mutation on activity and assembly of AsdP ......................... 118ppendix B. The detail dissection from monomer to dodecamer of AsdA ...................... 119ppendix C. The optimal pH and temperature of AsdA .................................................... 120ppendix D. The enzyme kinetics performed on pH 7.4 condition ................................. 121ppendix E. The assembly and cofactor list of other decarboxylase ............................. 122ppendix F. The tetrahedron assembly dodecamer structure proteins .......................... 123ppendix G. The membrane associated character of AsdA ............................................ 124ppendix H. The TEM of AsdA in different pH ................................................................... 125ppendix I. The confocal image of anti-His Ab labeled AsdA of E.coli ........................... 126ppendix J. The proposed inhibitor – AsdA complex ........................................................ 12