Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants

Abstract

The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane. Bacteria were chosen to include penicillin and cephalosporin 13-lactam producers and nonproducers. No significant amount of glutathione was found in any of the streptomycetes. Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17. Those streptomycetes that make B-lactam antibiotics also produce significant amounts of 8-(L-aaminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis. In Streptomyces clavuligerus, a potent producer of fl-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production. These and other observations indicate that ACV does not function as a protective thiol in streptomycetes. U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV. Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids. This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus. A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes.Numerous species of Streptomyces and one known species of Nocardia synthesize sulfur-containing penicillin, cephalosporin, and cephamycin antibiotics (2). Early investigators of the biosyntheses of these P-lactam compounds noticed the close structural relationship (28) between a key intermediate in the pathway, b-(L-oa-aminoadipyl)-L-cysteinyl-D-valine (ACV) and reduced glutathione, y-L-glutamyl-L-cysteinyl-glycine (GSH) (Fig. 1). GSH is the predominant thiol in eucaryotic organisms, in which it plays an important role in protecting cells against oxygen toxicity (17). It is found in most gram-negative bacteria but has not been detected in several major classes of gram-positive bacteria (13,14,26). Conflicting results were reported for the presence of GSH in streptomycetes. In one study, GSH could not be detected in cell extracts of Streptomyces griseus (14).In another study, low levels of GSH were found in Streptomyces lactamdurans (syn. Nocardia lactamdurans) (9). One purpose of the present study was to establish whether streptomycetes are able to produce GSH. Another was to determine whether ACV or other low-molecular-weight thiols play an antioxidant role in streptomycetes similar to that of GSH in other organisms. yeast extract (Difco), and 0.5% malt extract (Difco). Trypticase soy broth (TSB; BBL Becton Dickinson Microbiology Systems) was chosen for growth of these strains in liquid culture since it ...

Using Lactococcus lactis for glutathione overproduction

“…However, genes with similarity to gshB have until now not been observed in Gram-positives (see also Copley and Dhillon 2002). The assignment of a function to the corresponding γ-GCS-like hypothetical proteins is uncertain, particularly because these organisms are not known to synthesise GSH (Newton et al 1993(Newton et al , 1996. These observations do suggest that Gram-positive bacteria generally lack the capability to synthesise glutathione, although some strains are able to import glutathione from the environment (Sherrill and Fahey 1998).…”

The Substrate-induced Conformational Change of Mycobacterium tuberculosis Mycothiol Synthase

“…Members of the bacterial prokaryotic group termed actinomycetes lack the low molecular weight thiol, glutathione, typically found in many bacteria and in eukaryotes and instead produce mycothiol (acetyl-Lcysteine-1-D-myo-inosityl-2-deoxy-D-glucopyranoside) (1,2). Similar to glutathione, mycothiol provides the cell protection from oxidative damage and electrophilic toxins (3,4) and is maintained in the reduced state by a reductase (5).…”

“…Mycothiol biosynthesis in M. tuberculosis is a multistep process involving four enzymatic reactions catalyzed by MshA, MshB, MshC, and MshD (Scheme 1). Mycothiol synthase (MshD), 2 the last enzyme in mycothiol biosynthesis, catalyzes the acetylation of the cysteinyl amine of 1-D-myo-inosityl-2-L-cysteinylamido-2-deoxy-␣-D-glucopyranoside (desacetylmycothiol (DAM)) (9). The close proximity of the relatively reactive primary amine to the active thiol would potentially yield side reactions during mycothiol reduction and conjugation reactions, thus preventing mycothiol regeneration by mycothione reductase or mycothiol S-conjugate amidase.…”

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes

“…Because ACV structurally resembles glutathione, y-glutamyl-cysteinyl-glycine, the most common intracellular lowmolecular-weight thiol, we reasoned that the S. clavuligerus disulfide reductase might be related to glutathione reductase. However, extensive analysis of cell extracts of streptomycetes revealed that they lack glutathione (30) and glutathione reductase (2). Moreover, biochemical characterization of the S. clavuligerus reductase showed that, in contrast to glutathione reductase, it reduces a wide range of disulfides in low-molecular-weight compounds, is a potent reductant of disulfide bonds in proteins, and is composed of two nonidentical polypeptides.…”