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Assoc Prof Daan J Steenkamp Medical Scientist Tel 021-406-6105 Room 6.14
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Vuyo Mavumengwana PhD student    
Nick Watermeyer PhD student    

The Biochemistry of Pathogens: The metabolism and function of mycothiol in the mycobacteria.

The chemotherapy of pathogenic organisms relies on metabolic differences between pathogens and their mammalian hosts. Such a difference came to light in 1994 with the discovery and determination of the structure of mycothiol in our laboratory. Mycothiol is the principal low molecular mass thiol in the Actinomycetes, which include pathogens such as Mycobacterium tuberculosis. By contrast gram negative bacteria and practically all eukaryotes produce glutathione. These thiol compounds play a role in maintaining thiol groups, required for the activity of many enzymes, in a reduced state, and serve as cofactors for a number of enzymes involved in the detoxification and export of toxic compounds from cells.


Scheme 1

Scheme 1


We subsequently established essential features of the mycothiol biosynthetic pathway by showing that α-D-glucoaminyl-inositol (α-D-GI) is an intermediate of the pathway, which is then ligated to cysteine to produce 1-D-myo-inosityl-2-(L-cysteinyl)-amino-2-deoxy-α-D-glucopyranoside (des-acetylmycothiol). Mycothiol is finally produced by acetylation with acetyl-SCoA as the acetate donor. The earlier steps in the pathway were more recently elucidated by research groups in San Diego and Vancouver.

Mycothiol plays a role in the detoxification of some harmful compounds, such as alkylating agents, in a two-step process involving non-enzymatic alkylation of mycothiol to produce alkylated mycothiol, MSX in Scheme 2, which is then enzymatically cleaved into a mercapturic acid  and α-D-GI. Mycothiol also serves as a cofactor of the following enzymes:

  • A bifunctional enzyme formaldehyde dehydrogenase / S-nitrosomycothiol reductase
  • presumably also of a mycobacterial glyoxalase I and II, for the detoxification of methylglyoxal.
  • Maleyl-puruvate isomerase

This list may be expected to increase.


Scheme 2: Biosynthesis and some ancillary reactions involving mycothiol:
Glossary:  mshA: term for two reactions catalyzed by a glycosyltransferase and phosphatase, mshB: N-acetyl-α-D-GI-N-deacetylase, mshC: α-D-GI-L-cysteine-ligase, mshD: des-acetylmycothiolacetylase, mtr: mycothiol reductase,  mca: mycothiol-S-conjugate lyase, X: alkylating agent.

Disruption of the glycosyltransferase or mshC gene in Mycobacterium tuberculosis resulted in cells that were unable to grow in culture. Mycothiol biosynthesis can, therefore, be regarded as essential for the survival of M.tuberculosis . In M.tuberculosis mycothiol levels increase several-fold  when the bacteria are cultured into stationary phase, and it is, therefore, possible that mycothiol  plays a role in the attainment of a state of dormancy. The dormant state is thought to allow longterm survival of mycobacteria within their hosts. In this state the cells are unaffected by antibiotics and subsequent reactivation, when treatment had been discontinued, can result in a further episode of active tuberculosis.


Mycothiol biosynthesis as a drug target:

Since there is no mammalian counterpart to the pathway it should be possible to achieve a selective inhibition of mycothiol biosynthesis.  We are continuing with studies to test synthetically accessible α-D-GI analogues as substrates or inhibitors of these enzymes. Towards this end a number of α-D-GI analogues in which the inositol moiety is replaced with symmetrical aglycons have been synthesized by our collaborators in the Departments of Chemistry at UCT and at Rutgers University in the USA. At least some of these can serve as substrates of mshB and mca. An additional thrust of the synthetic work had been  to produce analogues of N-acetyl-α-D-GI in which the amide linkage had been replaced with an isostere having tetrahedral configuration, thus mimicking the transition state for cleavage of the amide bond by either mshB or mca.

MshC catalyzes the synthesis of desacetyl-mycothiol with adenylyl-cysteine as an activated intermediate, since this enzyme is a homolog of  the aminoacyl-tRNA-synthetases:
Scheme 3: Proposed  mechanism of ligation of α-D-GI to cysteine:

In the above scheme coordination of the sulphur atom of cysteine to zinc provides specificity of the enzyme for cysteine, and mshC is therefore a zinc metalloenzyme. Inhibition of the enzyme by transition state analogs presents a different problem from that encountered in the synthesis of γ-glutamylcysteine, the first step of glutathione biosynthesis, where the activated intermediate is a γ-glutamylphophoric acid anhydride. Very effective inhibitors, such as buthioninesulfoximine, were synthesized as mimics of the tetrahedral transition state  of γ-glutamylcysteine synthetase. It remains to be seen whether transition state analogs lacking the adenylate moiety of the probable mshC transition state are effective inhibitors of mshC.

An alternate strategy currently being explored is to produce subversive substrates of mycothiol reductase, an enzyme with a similar function to that of glutathione reductase. In such compounds analogues of α-D-GI are attached to naphthoquinones via N-alkyl chains of varying length. Naphthoquinones can accept single electrons directly from the flavocoenzyme in disulfide reductases and transfer them to oxygen with the resultant formation of toxic reactive oxygen intermediates.  Hopefully the presence of the pseudodisaccharide moiety will confer specificity for redox cycling with mycothiol reductase, rather than with enzymes present in the host.



On the isolation of mycothiol:

Steenkamp DJ, Vogt RN. (2004) Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols. Anal Biochem 325, 21-7.

On the structure of mycothiol:

Spies HSC and Steenkamp, DJ (1994) Novel thiols of intracellular pathogens: Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis. Eur J. Biochem. 224, 203 – 213.

On the mycothiol biosynthetic pathway:

Bornemann, C. , Jardine, M.A., Spies, H.S.C. and Steenkamp*, D.J. (1997) The biosynthesis of mycothiol: elucidation of the sequence of steps in Mycobacterium smegmatis. Biochem.J. 325, 623 - 629.

Newton GL, Ta P, Bzemek KP and Fahey RC (2006) The biochemistry of the early steps of mycothiol synthesis. J.Biol.Chem. 281, 33910 – 20.

On the role of mycothiol in detoxification:

Newton GL, Av-Gay Y and Fahey RC (2000) A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol-S-conjugate amidase.
Biochemistry 39, 10739 – 46.

Buchmeier NA, Newton GL, Koledin T, Fahey RC. (2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol. 47, 1723-32.

Vogt RN, Steenkamp DJ, Zheng R, Blanchard JS. (2003) The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase. Biochem J. 374, 657-66.


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