Abstract
Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe(2+) > Co(2+) > Zn(2+) > Mn(2+) and Ni(2+). Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe(2+) under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe(2+) and Zn(2+) under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe(2+)-MshB and Zn(2+)-MshB.
Highlights
JUNE 10, 2011 VOLUME 286 NUMBER 23 ins [1,2,3,4]
The enzymes involved in MSH biosynthesis and detoxification (Fig. 1A), including the metalloenzymes N-acetyl-1-D-myo-inosityl-2amino-2-deoxy-␣-D-glucopyranoside deacetylase (MshB) and MSH-conjugate amidase, are targets for the development of antibiotics for the treatment of diseases such as tuberculosis [5,6,7,8,9,10]
One possible mechanism uses a single bifunctional general acid-base catalysis (GABC) to facilitate the hydrolysis of GlcNAc-Ins, whereas the other uses a GABC pair to carry out this reaction
Summary
JUNE 10, 2011 VOLUME 286 NUMBER 23 ins [1,2,3,4]. MSH is likely to be critical for the survival of mycobacteria inside activated macrophages, where the mycobacteria are subjected to oxidative bursts. There have been several examples over the last decade of Fe2ϩ-enzymes being misidentified as exclusive Zn2ϩ-enzymes, including peptide deformylase, S-ribosylhomocysteinase (LuxS), UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC), and possibly histone deacetylase-8 (HDAC8) (20 –27) In all these enzymes, the Fe2ϩ cofactor is either exclusively preferred or is preferred over Zn2ϩ under certain environmental conditions. MshB exhibits a higher affinity for Zn2ϩ over Fe2ϩ, it is likely that higher free [Fe2ϩ] accounts for the cofactor preferences that are observed in these experiments. These results suggest that MshB likely uses Fe2ϩ and Zn2ϩ as biological cofactors under different environmental conditions. These results may have important biological implications in light of the dynamic changes in zinc and iron concentrations that occur during the course of infection and suggest that biologically effective inhibitors will need to dually target Fe2ϩ-MshB and Zn2ϩ-MshB
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