Characterization of glutamate-cysteine ligase and glutathione synthetase from the δ-proteobacterium Myxococcus xanthus.

Glutathione (GSH) is synthesized in two ATP-dependent reactions by glutamate-cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram-negative bacterium belonging to δ-proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+ , but not by Mg2+ , and stabilized in the presence of 5 mM Mn2+ or Mg2+ . Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki  = 2.1 µM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+ . MxGs was inhibited by GSSG at Ki  = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.