Metabolomics Analysis Identifies D-Alanine-D-alanine Ligase as the Primary Lethal Target of D-cycloserine in Mycobacteria

Authors

Steven M. Halouska, University of Nebraska-LincolnFollow
Robert J. Fenton, University of Nebraska - LincolnFollow
Denise K. Zinniel, University of Nebraska - LincolnFollow
Darrell D. Marshall, University of Nebraska-Lincoln
Raul G. Barletta, University of Nebraska - LincolnFollow
Robert Powers, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2014

Citation

J Proteome Res. 2014 February 7; 13(2): 1065–1076. doi:10.1021/pr4010579.

Comments

© 2013 American Chemical Society. Used by permission.

Abstract

D-cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR)- drug resistant strains of Mycobacterium tuberculosis. D-cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of Alanine racemase (Alr) and D-Alanine-D-alanine ligase (Ddl). Although, the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of D-alanyl-Dalanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of D-alanine thus allowing DCS to outcompete D-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce D-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.