Structure, Assembly, and Mechanism of a PLP-dependent Dodecameric L-Aspartate β-Decarboxylase
Date Issued
2009
Date
2009
Author(s)
Chen, Hui-Ju
Abstract
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.
Subjects
beta-decarboxylase
aminotransferase
pyridoxal 5’-phosphate
pH dependent
bi-functional enzyme
X-ray diffraction
dodecamer assembly
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