Sunil V. Sharma

Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Chemical and chemoenzymatic syntheses of bacillithiol: a unique low-molecular-weight thiol amongst low G + C Gram-positive bacteria

In eukaryotes and Gram-negative bacteria, the cysteinyl tripeptide glutathione (GSH, Figure 1) is the predominant low-molecular-weight thiol. It plays a critical role in maintaining an intracellular reducing environment and serves many other important metabolic functions.⁽¹⁾ For instance, the reversible formation of GS-S-protein disulfides (glutathionylation) is an important post-translational modification for regulating protein function and protecting exposed cysteine residues from irreversible oxidative damage.⁽²⁾ Glutathione-Stransferases also mediate xenobiotic detoxification by S conjugation with GSH. Most Gram-positive bacteria lack GSH, but instead produce other, distinctly different low-molecular weight thiols. Gram-positive high G+C content actinobacteria produce mycothiol (MSH, Figure 1), which serves analogous functions to GSH.⁽³⁾ Low G+C Gram-positive bacteria (Firmicutes) produce neither GSH nor MSH and until recently the identity of their major, cysteine-derived, low-molecular-weight thiol has been elusive. In 2007, an unknown 398 Da thiol was observed in Bacillus anthracis cell extracts⁽⁴⁾ and in Bacillus subtilis as a mixed disulfide with the redox controlled ohr regulator protein (OhrR).⁽⁵⁾ The same thiol was subsequently isolated by treating Deinococcus radiodurans cell extracts with monobromobimane (mBBr) from which the structure of bacillithiol (BSH, 1) was then elucidated as its corresponding fluorescently labeled Sbimane (mB) derivative BSmB (3, Figure 1).⁽⁶⁾

Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low‐molecular‐weight (LMW) thiol in many low‐G+C Gram‐positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa, redox potential and thiol–disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM, which is significantly greater than previously observed from single measurements taken during mid‐exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.

α-Amino acid Tröger base derivatives, possible conformationally restricted scaffolds?

The first synthesis of innovative alpha-amino acid conjugates of Tröger base is reported; their potential application as conformationally restricted scaffolds is proposed and has been investigated using high level ab initio calculations.

Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways

BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²⁺-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities.

An expedient one-pot synthesis of para-tert-butylcalix[8]- and [9]arene

We report the first efficient synthesis of para-tert-butylcalix[8]arene and -[9]arene via an exceptionally straightforward innovative protocol that takes place at ambient temperature, employing readily available tin(IV) chloride and s-trioxane.

Thiol Redox and pKa Properties of Mycothiol, the Predominant Low-Molecular-Weight Thiol Cofactor in the Actinomycetes.

The thiol pKa and standard redox potential of mycothiol, the major low‐molecular‐weight thiol cofactor in the actinomycetes, are reported. The measured standard redox potential reveals substantial discrepancies in one or more of the other previously measured intracellular parameters that are relevant to mycothiol redox biochemistry.

Hybrid calix[4]arenes via ionic hydrogenation and transition-metal-mediated processes.

We report the first application of ionic hydrogenation for the synthesis of upper-rim urea- or carbamate-derived hybrid calix[4]arenes. Subsequent metal-mediated transformations using 4-iodophenylurea calixarenes afforded structurally unique 1,3-di(biaryl)-, 1,3-di(biarylalkyne)-, or 1,3-(biaryl)(biarylalkyne)-derived hybrid calixarenes.

Ferrocenium salts mediate para-tert-butylcalixarene synthesis

Ferrocenium salts mediate high yielding one-pot and convergent syntheses of para-tert-butylcalixarenes in mild non-Lewis or Brønsted acidic reaction conditions; EPR indicates complex formation between the s-trioxane and the ferrocenium salt.

N-methyl-bacillithiol, a new metabolite discovered in the Chlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed.

Low-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed that Chlorobaculum tepidum contained a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, the Cba. tepidum thiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in all Chlorobi examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobi, Bacteroidetes, Deinococcus-Thermus, and Firmicutes, but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Significance Statement Here, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor in Cba. tepidum and the gene, nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailed in vivo and in vitro dissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.