Thiols Screened by the Neocarzinostatin Protein for Preserving or Detoxifying its Bound Enediyne Antibiotic

Abstract

Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne‐containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site‐directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral‐charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol‐dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein‐bound chromophore. The anionic side‐chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol‐screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge–charge repulsion in the outer‐cell environment and detoxifying the toxin in the inner‐cell body for self‐resistance by accepting the cell‐produced neutral mycothiol.

Comparison of Antibiotic Resistance Mechanisms in Antibiotic-Producing and Pathogenic Bacteria

“…There are two self-resistance-related genes, ncsA encoding apoprotein for the sequestration of the enediyne chromophore, and ncsA1 encoding efflux pump transporter within the cluster [412]. In addition, mycothiol-dependent detoxication may be involved in the self-resistance [413]. C-1027 is a chromoprotein antitumor antibiotic produced by S. globisporus and composed of an apoprotein and the enediyne chromophore.…”

Comparison of Strategies to Overcome Drug Resistance: Learning from Various Kingdoms

“…The amino acid residues D33 and D99 of the apoprotein play significant roles for self-protection. In addition, the neocarzinostatn carrier protein and mycothiol-dependent cellular detoxication are also important [ 503 ]. Calicheamicin is the non-chromoprotein enediyne type of antitumor antibiotic that is produced by Micromonospora echinospora .…”