Identification of the Molecular Features that Facilitate the Thermal Adaptation of Aminoglycoside O-Nucleotidlytransferase 4' and a Novel Kinetic Mechanism for Nucleotidyl Transfer

The aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that detoxifies many aminoglycoside antibiotics by nucleotidylating them at the C4'-OH site. Two variants with increased thermostability (T130K, D80Y), which differed from the mesophilic wild type ANT (WT) only by a single residue, showed contrasting ligand binding thermodynamics properties. While T130K showed identical properties to WT, D80Y had stark differences. Our thermodynamics studies demonstrated that solvent reorganization becomes the major contributor to ligand binding as the temperature increases for WT and T130K, while changes in protein dynamics is the main contributor for D80Y. Our structural studies have shown that, in the T130K variant, there is an additional H-bond formed between the side chain of K130 and the backbone oxygen of S90, which explains the increased structural stability of the T130K. Despite having the highest melting temperature, an obvious additional stabilizing interaction is not identified in the D80Y structure. These observations have led us to further distinguish T130K from D80Y. We have concluded that T130K is the “thermostable variant”, since it has an increased structural stability yet mimics the ligand binding and protein dynamics behaviors of the mesophilic WT. Furthermore, we refer to D80Y as the “thermophilic variant”, since it behaves very differently from the WT in order to adapt to a harsher environment with elevated temperatures compared to that of the WT. Overall, we have shown that global thermodynamics of ligand-protein interactions and solvent effects may be among the molecular parameters that define true thermophilic enzyme behavior. The applicability of these findings to other systems remains to be seen and can be helpful in understanding if there is a general strategy that enzymes use to adapt to hot environments. We have also identified the catalytic mechanism of ANT. We propose a low barrier hydrogen bond (LBHB) formed between E52 and the ligand which enables the enzyme to bind to the ligand tightly. This LBHB is disrupted after the cofactor ATP is bound and the catalysis takes place, leading to the release of the modified aminoglycoside. LBHB thereby serves a novel “catch and release” mechanism for nucleotidyl transfer.