N -Acetyl-1- d - myo -Inosityl-2-Amino-2-Deoxy-α- d -Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis

Abstract

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-␣-Dglucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis. In the present study we found that a protein encoded by the M. tuberculosis open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M. smegmatis at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of M. smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the M. tuberculosis genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.Most gram-positive bacteria, including many strict aerobes, do not produce glutathione (7, 13), a key component of cellular mechanisms for protection against oxygen toxicity (5). In the search for other thiols in these organisms that might function like glutathione, a major thiol in streptomycetes was discovered (14) and later identified (12,19,21) as a novel conjugate of N-acetylcysteine (AcCys) and 1-D-myo-inosityl-2-amino-2-deoxy-␣-D-glucopyranoside (GlcN-Ins) producing mycothiol (MSH or AcCys-GlcN-Ins). Bornemann et al. (3) showed that chemically synthesized GlcN-Ins could be converted to CysGlcN-Ins and MSH by Mycobacterium smegmatis cell extracts in the presence of ATP, Mg 2ϩ , Cys, and acetate or acetyl coenzyme A (acetyl-CoA). They proposed that the final steps of MSH biosynthesis involve ATP-dependent ligation of Cys with GlcN-Ins, followed by acetylation via an acetyltransferase reaction involving acetyl-CoA. Measurements of GlcN-Ins and Cys-GlcN-Ins levels in M. smegmatis (1) and isolation of mutants defective in GlcN-Ins or Cys-GlcN-Ins production support the existence of this pathway (15).The earlier studies left undefined the biochemical reactions involved in GlcN-Ins formation. By analogy with the established biochemistry for production of the glycosylphosphatidylinositol (GPI) anchor, which contains as a component an isomer of GlcN-Ins with an ␣(1-6) linkage, one might expect that GlcN-Ins is produced by deacetylati...

Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein

“…Three homologs are found in Streptomyces coelicolor, and two homologs are found in Streptomyces avermitilis. The M. tuberculosis genome also has two paralogs, Rv1170, which codes for MshB (3,11,15,22), and Rv0323c, a gene that is present only in pathogenic mycobacteria and thus is absent in nonpathogenic M. smegmatis.…”

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

“…oside (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)(13)(14)(15).…”

Differential Expression of Mycothiol Pathway Genes: Are they Affected by Antituberculosis Drugs?

“…Oligonucleotides were allowed to enter the mycobacteria through passive diffusion and mycobacterial growth (GI) was monitored every 24 h. A control oligonucleotide, not matching any ORF in the M. tuberculosis database was used to control for the effect of phosphorothioate linked DNA on mycobacterial growth. product under normal culture conditions (9). It was therefore suggested that mycothiol can be produced on demand from the large substrate pool, however, control over the activity of the deacetylase has not been shown previously (9).…”

“…No differences in expression levels of Rv1170 were observed over the time range of exposure to INH suggesting that, once the gene is induced, expression remains at a constant level in the constant presence of the inducer. This enzyme is thought to be the control point for mycothiol biosynthesis (9). There is a substantial pool of its substrate present, but low levels of Figure 4.…”

“…Mycothiol has been shown to be a major thiol found in, and unique to, some mycobacteria (Actinomycetes) (3,8) and is currently considered to play an analogous role to that of GSH in the mycobacterial defence against reactive oxygen toxicity. By means of comparative genomics, the first enzyme shown to be involved in this pathway, GlcNAc-Ins Deacetylase (Rv1170), was recently identified, cloned and characterized (9). It was shown that mycothiol is synthesized by GlcNAc-Ins deacetylase and that this enzyme constitutes the control point of mycothiol synthesis.…”