Daptomycin is an antibiotic frequently used as drug of last resort against Gram-positive Multi-Drug Resistant pathogens such as vancomycin-resistant enterococci. Unsurprisingly, as the use of daptomycin has increased, the number of pathogens evolving resistance has increased as well. Inhibition of the major antibiotic stress pathway (LiaFSR) responsible for daptomycin resistance has been suggested as a strategy to retain the efficacy of daptomycin. Such a strategy was shown to be effective in vitro, however alternative pathways to resistance were identified, including some with adaptive mutations in MurAA (MurAAA149E). MurAA catalyzes the first committed step in peptidoglycan biosynthesis. Cells expressing MurAAA149E has increased susceptibility to glycosyl hydrolases consistent with decreased cell wall integrity. High-resolution structures of MurAA in complex with the inhibitor, fosfomycin, and substrate, UDP-N-acetylglucosamine along with steady-state substrate kinetics, substrate and inhibitor binding assays indicated a modest decrease in MurAAA149E activity. Interestingly, MurAAA149E had a 16-fold increase in affinity for MurG. MurG performs the last intracellular step of peptidoglycan synthesis. Daptomycin attack leads to mislocalization of critical cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG localize at division septa and thus MurAAA149E may contribute to cellular fitness during DAP exposure by increasing the stability of MurAA-MurG interactions at the division septa, but in doing so decreases cell resistance to attack by hydrolases. Our findings suggest that an adaptive strategy to circumvent LiaFSR inhibition through changes in MurAA may be ineffective in vivo as cellular hydrolases are an important component of the host response to pathogens.