An <Emphasis Type="Italic">N</Emphasis>-acyl homolog of mycothiol is produced in marine actinomycetes

Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N-propionyl-desacetyl-mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol, were found in all strains. High levels of mycothiol S-conjugate amidase activity, a key enzyme in mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that mycothiol-dependent detoxification is important at this developmental stage.     

Purification and characterization of Mycobacterium tuberculosis 1D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase, MshB, a mycothiol biosynthetic enzyme

Mycothiol (MSH, AcCys-GlcN-Ins) is the major low molecular weight thiol in actinomycetes and is essential for growth of Mycobacterium tuberculosis. MshB, the GlcNAc-Ins deacetylase, is a key enzyme in MSH biosynthesis. MshB from M. tuberculosis was cloned, expressed, purified, and its properties characterized. Values of k(cat) and K(m) for MshB were determined for the biological substrate, GlcNAc-Ins, and several other good substrates. The substrate specificity of MshB was compared to that of M. tuberculosis mycothiol S-conjugate amidase (Mca), a homologous enzyme having weak GlcNAc-Ins deacetylase activity. Both enzymes are metalloamidases with overlapping amidase activity toward mycothiol S-conjugates (AcCySR-GlcN-Ins). The Ins residue and hydrophobic R groups enhance the activity with both MshB and Mca, but changes in the acyl group attached to GlcN have opposite effects on the two enzymes.     

A coupled spectrophotometric assay for l-cysteine:1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase and its application for inhibitor screening

Most actinomycetes, including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PP(i). MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2,024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC(50) approximately 100 microM.     

Unusual production of glutathione in <Emphasis Type="Italic">Actinobacteria</Emphasis>

Most Actinobacteria produce mycothiol as the major thiol. In addition to mycothiol Rhodococcus AD45 generates a substantial level of glutathione possibly using genes acquired in a lateral transfer. Instead of mycothiol, Rubrobacter radiotolerans and Rubrobacter xylanophilus produce glutathione, whose synthesis appears to involve enzymes substantially different from those in other organisms.     

Identification and characterization of a diamide sensitive mutant of Mycobacterium smegmatis

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.     

Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.     

A fluorescence-based assay for measuring N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase activity

Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase) activity. The current assay for measuring MshB activity requires the fluorescent labeling of reaction mixtures and subsequent analysis using high-performance liquid chromatography (HPLC), resulting in a significant amount of processing time per sample. Here we describe a more rapid fluorescnce-based assay for the measurement of MshB activity that does not require HPLC analysis and can be carried out in multiwell plates. This fluorescamine (FSA)-based assay was used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine (GlcNAc) by MshB, and the results from these experiments support the hypothesis that the inositol moiety primarily contributes to the affinity of GlcNAc–Ins (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside) for MshB. The rapid nature of this assay will aid efforts toward a more detailed biochemical characterization of MshB. Furthermore, because this assay relies on the formation of a primary amine, it could be adapted to measure the activity of mycothiol-S-conjugate amidase, a metal-dependent amidase that is a potential drug target involved in the mycothiol detoxification pathway.     

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum Dimerizes at High Protein Concentration and Exclusively Receives Electrons from Thioredoxin Reductase

NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10⁶ and 5.6 × 10⁶ m⁻¹ min⁻¹, respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pK_a lower than 6.5 for nucleophilic attack, whereas the pK_a of the C-terminal cysteine is ∼10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.     

Glutathione analogs in prokaryotes

Background

Oxygen is both essential and toxic to all forms of aerobic life and the chemical versatility and reactivity of thiols play a key role in both aspects. Cysteine thiol groups have key catalytic functions in enzymes but are readily damaged by reactive oxygen species (ROS). Low-molecular-weight thiols provide protective buffers against the hazards of ROS toxicity. Glutathione is the small protective thiol in nearly all eukaryotes but in prokaryotes the situation is far more complex.

Scope of review

This review provides an introduction to the diversity of low-molecular-weight thiol protective systems in bacteria. The topics covered include the limitations of cysteine as a protector, the multiple origins and distribution of glutathione biosynthesis, mycothiol biosynthesis and function in Actinobacteria, recent discoveries involving bacillithiol found in Firmicutes, new insights on the biosynthesis and distribution of ergothioneine, and the potential protective roles played by coenzyme A and other thiols.

Major conclusions

Bacteria have evolved a diverse collection of low-molecular-weight protective thiols to deal with oxygen toxicity and environmental challenges. Our understanding of how many of these thiols are produced and utilized is still at an early stage.

General significance

Extensive diversity existed among prokaryotes prior to evolution of the cyanobacteria and the development of an oxidizing atmosphere. Bacteria that managed to adapt to life under oxygen evolved, or acquired, the ability to produce a variety of small thiols for protection against the hazards of aerobic metabolism. Many pathogenic prokaryotes depend upon novel thiol protection systems that may provide targets for new antibacterial agents. This article is part of a Special Issue entitled Cellular functions of glutathione.     

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

Actinomycetes are a group of gram-positive bacteria that includes pathogenic mycobacterial species, such as Mycobacterium tuberculosis. These organisms do not have glutathione and instead utilize the small molecule mycothiol (MSH) as their primary reducing agent and for the detoxification of xenobiotics. Due to these important functions, enzymes involved in MSH biosynthesis and MSH-dependent detoxification are targets for drug development. The metal-dependent deacetylase 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 in MSH biosynthesis. Herein we examine the chemical mechanism of MshB. We demonstrate that the side chains of Asp-15, Tyr-142, His-144, and Asp-146 are important for catalytic activity. We show that NaF is an uncompetitive inhibitor of MshB, consistent with a metal-water/hydroxide functioning as the reactive nucleophile in the catalytic mechanism. We have previously shown that MshB activity has a bell-shaped dependence on pH with pK(a) values of ～7.3 and 10.5 (Huang, X., Kocabas, E. and Hernick, M. (2011) J. Biol. Chem. 286, 20275-20282). Mutagenesis experiments indicate that the observed pK(a) values reflect ionization of Asp-15 and Tyr-142, respectively. Together, findings from our studies suggest that MshB functions through a general acid-base pair mechanism with the side chain of Asp-15 functioning as the general base catalyst and His-144 serving as the general acid catalyst, whereas the side chain of Tyr-142 probably assists in polarizing substrate/stabilizing the oxyanion intermediate. Additionally, our results indicate that Tyr-142 is a dynamic side chain that plays key roles in catalysis, modulating substrate binding, chemistry, and product release.