Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme.
Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.
Department of Chemistry & Chemical Biology and the Center for Integrative Proteomics, Rutgers University, Piscataway, NJ 08854, USA.
Brahm J
BJ
Yachnin
Department of Chemistry & Chemical Biology and the Center for Integrative Proteomics, Rutgers University, Piscataway, NJ 08854, USA.
Sagar D
SD
Khare
D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D009154:Mutation; D011623:gamma-Glutamyl Hydrolase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D004792:Enzyme Precursors; D011623:gamma-Glutamyl Hydrolase
Acy1 M20 metallopeptidases family, carboxypeptidase G2; circular permutation; directed enzyme-prodrug therapy; methotrexate