Optimizing Visualization of Aminoglycoside Acetyltransferase Mutant Enzyme Activity onto 3D Protein Structures to Determine "Patches" of Functional Importance
Damon Holt, Tran Hoang Danh Nguyen, Alejandra Portillo, Gabriela Tozlovan, Hazel N. Leia Martel
Department of Chemistry & Biochemistry
Faculty Supervisor: Misty L. Kuhn
Identifying amino acids in proteins that are critical for enzyme activity and substrate specificity is important because these residues control how an enzyme binds to its substrates and catalyzes its reaction. In this study, we investigated the AAC(6')-Ig aminoglycoside acetyltransferase enzyme, which acetylates aminoglycoside antibiotics and aids antibiotic resistance in clinical settings. To determine which residues were important for substrate recognition and acetylation, we examined the enzyme crystal structure and selected amino acids within the active site and substrate binding pockets that might interact with antibiotics or participate in catalysis. We generated 61 single amino acid substitutions and assessed their effects using antibiotic disc susceptibility tests toward three aminoglycosides. We also purified and kinetically screen all soluble proteins for activity. To manage these large datasets, we categorized activity into low, medium, and high thresholds and mapped the results onto the crystal structure to identify critical regions for activity. Although some key residues overlapped across antibiotic substrates, many important residues were located outside of the active site, indicating that broader regions of the protein contribute to enzyme activity. These findings highlight the complexity of determining how an enzyme functions and predict which residues will contribute to activity.
This study was funded in part by the National Institutes of Health Department of Health and Human Services, National Institute of General Medical Sciences under Award Number R35GM133506 (to MLK). Additional funding was provided in part by The Camille and Henry Dreyfus Foundation under Award Number TH-22-104 (to MLK).