The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Abstract

2 as their primary reducing agent and in xenobiotic metabolism for the detoxification of drugs and other toxins (1-4). MSH is likely to be critical for the survival of mycobacteria inside activated macrophages, where the mycobacteria are subjected to oxidative bursts. Consequently, the enzymes involved in MSH biosynthesis and detoxification (Fig. 1A), including the metalloenzymes N-acetyl-1-D-myo-inosityl-2-amino-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 (5-10).The enzyme MshB catalyzes the hydrolysis of (GlcN-Ins) and acetate, the fourth overall step in MSH biosynthesis (rate-limiting step) (11). MshB is an attractive drug target because it is a metalloenzyme; there are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensinconverting enzyme (12-15). Inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion. Consequently, a comprehensive understanding of metalloenzyme cofactor preferences is necessary for the development of potent and specific metalloenzyme inhibitors.MshB was previously identified as a Zn 2ϩ -dependent enzyme based on the observations that the enzyme copurifies with Zn 2ϩ (Fig. 1B) and that the enzyme activity is reversibly inhibited by treatment with 1,10-phenanthroline (16 -18). On the basis of the structure of the enzyme active site, MshB is thought to catalyze the hydrolysis of GlcNAc-Ins via one of two potential chemical mechanisms using general acid-base catalysis (GABC) (19). One possible mechanism uses a single bifunctional GABC to facilitate the hydrolysis of GlcNAc-Ins, whereas the other uses a GABC pair to carry out this reaction. However, Fe 2ϩ was not examined as a potential cofactor in these experiments. Furthermore, MshB was purified using zinc immobilized metal ion affinity chromatography (IMAC) under aerobic conditions, which is biased toward zinc incorporation into metalloenzymes (16). Purified MshB contains nickel (0.82 eq) when purified using nickel IMAC (aerobic conditions) (16). There have been several examples over the last decade of Fe 2ϩ -enzymes being misidentified as exclusive Zn 2ϩ -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 Fe 2ϩ cofactor is either exclusively preferred or is preferred over Zn 2ϩ under certain environmental conditions. The

Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

“…MshB prefers Fe 2ϩ under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe 2ϩ and Zn 2ϩ under aerobic conditions as the metal content of the medium is altered. MshB has a bell-shaped dependence on pH (subsaturating concentrations of substrate, V/K), indicating that there are two ionizations that are important for maximal catalytic activity (14), consistent with a reaction that proceeds through either a single bifunctional general acid-base catalyst (GABC) or GABC pair mechanism (21).…”

“…Protein Expression and Purification-The previously reported plasmid encoding the MshB gene from Mycobacterium smegmatis containing an N-terminal His-MBP tag was used as the template for preparation of mutant plasmids (14). All mutant plasmids were prepared using the QuikChange Lightning site-directed mutagenesis kit (Stratagene).…”

“…1A). MshB is an attractive drug target because it catalyzes the rate-limiting step in MSH biosynthesis (11), it is a metalloenzyme (12)(13)(14), and the three-dimensional structure is known (15,16). There are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensin-converting enzyme (17)(18)(19)(20).…”

“…There are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensin-converting enzyme (17)(18)(19)(20). Because inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion, we previously examined the cofactor preferences of MshB and found that MshB is a cambialistic metalloenzyme whose in vitro activity follows the following trend: Fe 2ϩ Ͼ Co 2ϩ Ͼ Zn 2ϩ Ͼ Mn 2ϩ Ͼ Ni 2ϩ (14). Additionally, we found that the cofactor bound to MshB is dependent on environmental conditions (14).…”

“…Because inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion, we previously examined the cofactor preferences of MshB and found that MshB is a cambialistic metalloenzyme whose in vitro activity follows the following trend: Fe 2ϩ Ͼ Co 2ϩ Ͼ Zn 2ϩ Ͼ Mn 2ϩ Ͼ Ni 2ϩ (14). Additionally, we found that the cofactor bound to MshB is dependent on environmental conditions (14). MshB prefers Fe 2ϩ under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe 2ϩ and Zn 2ϩ under aerobic conditions as the metal content of the medium is altered.…”