Structural-Based Engineering for Transferases to Improve the Industrial Production of 5′-Nucleotides

The existence of a flexible region for substrate recognition by an enzyme was first discovered through a structure comparison study among ribonuclease T1–related molecules, including intact molecules, intact molecules complexed with 2′-guanylic acid (GMP), and carboxymethylated molecules complexed with 2′-GMP. Next, it was demonstrated that transglutaminase activity could be enhanced by identifying the flexible residues that affect the active site using nuclear magnetic resonance. Finally, such an approach was used to increase the production of 5′-inosinic acid (5′-IMP) and 5′-GMP. The enzyme is a nonspecific acid phosphatase with pyrophosphate-nucleoside phosphotransferase activity that is C-5′ position-selective. The crystal structure of G74D/I153T with a reduced KM is virtually identical to that of the wild type, and neither of the side chains that were introduced Asp74 and Thr153, directly interact with the nucleoside, although both residues are situated near a potential nucleoside binding site. In addition, the two regions around residues 70 and 140 have the most flexible conformational rearrangement upon phosphate analogue binding. On the basis of three-dimensional (3D) structural information, some practical, high-performance enzymes for 5′-nucleotides production were discovered, and new varieties of microorganisms were developed.