Sk. Nayak et al., Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin, BIOCHEM, 38(31), 1999, pp. 10052-10058
Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the
group of ribonucleolytic toxins called ribotoxins. It specifically cleaves
a single phosphodiester bond in a conserved stem and loop structure in the
28S rRNA of large ribosomal subunit and potently inhibits eukaryotic prote
in synthesis. Restrictocin contains 149 amino acid residues and includes fo
ur cysteines at positions 5, 75, 131, and 147. These cysteine residues are
involved in the formation of two disulfide bonds, one between Cys 5 and Cys
147 and another between Cys 75 and Cys 131. In the current study, all four
cysteine residues were changed to alanine individually and in different co
mbinations by site-directed mutagenesis so as to remove one or both the dis
ulfides. The mutants were expressed and purified from Escherichia coli. Rem
oval of any cysteine or any one of the disulfide bonds individually did not
affect the ability of the toxin to specifically cleave the 28S rRNA or to
inhibit protein synthesis in vitro. However, the toxin without both disulfi
de bonds completely lost both ribonucleolytic and protein synthesis inhibit
ion activities. The active mutants, containing only one disulfide bond, exh
ibited relatively high susceptibility to trypsin digestion. Thus, none of t
he four cysteine residues is directly involved in restrictocin catalysis; h
owever, the presence of any one of the two disulfide bonds is absolutely es
sential and sufficient to maintain the enzymatically active conformation of
restrictocin. For maintenance of the unique stability displayed by the nat
ive toxin, both disulfide bonds are required.