Figure 6

Structure of loop regions located at the A/D monomer interface and catalytic centers. The 2F_o-F_c electron density maps of NylC_p2 (a), NylC_p2-G¹²² (b), NylC_p2-Y¹³⁰ (c), NylC_p2-G¹²²Y¹³⁰ (d), and NylC_p2-G¹²²Y¹³⁰A³⁶Q²⁶³ (e) are shown. Left panel: amino acid residues in monomer A (Asp/Gly122, Glu126, and His/Tyr130) and monomer D (Asp/Ala36) are shown as stick models. Right panel: amino acid residues in monomer A (Thr132 and Leu137) and monomer D (Tyr112, Tyr146, and Phe148) are shown as stick models. In the thermostable enzymes (c–e), “loop 3” and α3 (containing Tyr130, Thr132, and Leu137) (in monomer A) are mutually stabilized by contacts with “loop 1” and “loop 4” (containing Tyr146 and Phe148) in monomer D. Leu137 (in monomer A) has contacts with monomer D at Tyr146, Phe148, and Tyr112 (in helix α1), suggesting that hydrophobic interactions around Leu137 stabilize the protein structure. D36A substitution in NylC_p2-G¹²²Y¹³⁰Q²⁶³ mutant (T_m = 84 °C) contributes to the increased stability, probably by the reduction of electrostatic repulsion between Asp36-COO⁻ (monomer D) and Glu126-COO⁻ (monomer A) (e).