Structure–Stability–Activity Relationship in Covalently Cross-linked N-Carbamoyl d-Amino acid Amidohydrolase and N-Acylamino acid Racemase

N-Acylamino acid racemase (NAAAR) and N-carbamoyl-d-amino-acid amidohydrolase (d-NCAase) are important biocatalysts for producing enantiopure α-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 °C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity.