Antibiotic resistance is a serious and growing threat to human health. The environmental microbiome is a rich reservoir of resistomes, offering opportunities to discover new antibiotic resistance genes. Here we demonstrate an integrative approach of utilizing gene sequence and protein structural information to characterize unidentified genes that are responsible for the resistance to the action of rifamycin antibiotic rifampin, a first-line antimicrobial agent to treat tuberculosis. Biochemical characterization of four environmental metagenomic proteins indicates that they are adenosine diphosphate (ADP)-ribosyltransferases and effective in the development of resistance to FDA-approved rifamycins. Our analysis suggests that even a single residue with low sequence conservation plays an important role in regulating the degrees of antibiotic resistance. In addition to advancing our understanding of antibiotic resistomes, this work demonstrates the importance of an integrative approach to discover new metagenomic genes and decipher their biochemical functions.