L-天門冬胺酸β-去羧酶十二倍體蛋白之立體功能結構鑑定及磷酸比哆醛依順機制

Abstract

The focus of this PhD thesis is the type-I PLP (pyridoxal 5’-phosphate) enzyme L-aspartate β-decarboxylase (ASD, from Alcaligenes faecalis) with particular reference to an analysis of protein structure determination and functional activity characterization. The ASD has bi-functional activity. The major one being the conversion of aspartate to alanine and CO2 by decarboxylation, but additionally, it also functions to transaminate aspartate to produce oxaloacetate. Similar to the homodimeric aminotransferases, its protein subunit comprises a large and a small domain, of 410 and 120 residues, respectively. The crystal structure reveals a dodecamer made of six identical dimers which are arranged in a truncated tetrahedron whose assembly involves tetramer and hexamer as intermediates. Based on this structure, we proposed a catalysis mechanism and four functional motifs: a substrate binding motif (βY-loop-βZ), a PLP binding site (Lys315), a regulatory motif (α1- α2 and α13- α15) and an assembly motif (α3- α5 and α16- α17). The additional helical motifs I (α3- α5) and II (α16- α17) participate in the oligomer formation. Triple mutations of S67R/Y68R/M69R or S67E/Y68E/M69E in motif I produced an inactive dimer. The functional dodecamer structure is rather distinct from the aminotransferase family. The PLP is bound covalently to Lys315 in the active site, while its phosphate group interacts with the neighboring Tyr134. Removal of the bulky side chain of Arg37, which overhangs the PLP group, improved the substrate affinity. Mutations in flexible regions produced the more active K17A and the completely inactive R487A. The structure also suggests that substrate binding triggers conformational changes essential for catalyzing the reaction. The substrate induced S-domain conformational change was elucidated by β-chloralanine–AsdP complex. Along the three-fold axis of ASD structure, there are four 1.4 A radius in size pores were appeared iv on each three α4 helices bundle of the plate shape hexamer. The surface electron potential of α4-α5 helices was changed from most positive charge to half hydrophobicity that cooperated with N-terminal moved and rotated about 5 A and 22.5 degree, respectively. The cross-interaction of S-domain involved with van der Waals force between α1 to α2, α1 to α20, and α20 to α21 helices those move together by hydrophobic patch I, II, and III. The Arg497 residue has observed that was contributed with stabilize carboxyl group of side chain of β-chloralanine-PLP complex as ATase’s enzymatic reaction.