title: Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase.
abstract: N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure alpha-amino acids. NAAAR forms an octameric assembly and displays induced fit movements upon substrate binding, while D-NCAase is a tetramer that does not change conformation in the presence of a ligand. To investigate the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes, cysteine residues predicted to form inter or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. The structures of A222C and A302C D-NCAases are nearly identical to the wild-type enzyme. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity.
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, HsinChu, Taiwan, 30013, ROC.
authors.forename: Wei-Chun
authors.initials: WC
authors.lastname: Chiu
authors.forename: Ji-Yu
authors.initials: JY
authors.lastname: You
authors.forename: Jai-Shin
authors.initials: JS
authors.lastname: Liu
authors.forename: Shih-Kuang
authors.initials: SK
authors.lastname: Hsu
authors.forename: Wen-Hwei
authors.initials: WH
authors.lastname: Hsu
authors.forename: Chien-Hua
authors.initials: CH
authors.lastname: Shih
authors.forename: Jenn-Kang
authors.initials: JK
authors.lastname: Hwang
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Wang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; D000591:Amino Acid Isomerases