Biochemical comparison of the Cu,Zn superoxide dismutases of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii

Abstract

Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and var. gattii. The N-terminal amino acid sequences of the two enzymes were similar, though not identical, and demonstrated homology with Cu,Zn SODs from other organisms. SOD activity was present in supernatants from stationary-phase cultures of isolates of C. neoformans var. neoformans and was also present from the mid-log phase onwards in cultures of an acapsular mutant of C. neoformans var. neoformans. SOD activity was practically undetectable in culture supernatants from isolates of C. neoformans var. gattii. The C. neoformans var. neoformans SOD had a reduced relative molecular mass of 19 kDa, and in its nonreduced form the enzyme was present as a 125-kDa species. Isoelectric focusing indicated that four species with pIs of 5.9, 6.15, 6.35, and 6.6 were present. The equivalent reduced molecular mass of the C. neoformans var. gattii enzyme was 19 kDa, with a single species present under nonreducing conditions (relative molecular mass of 145 kDa) with a pI of 7.5. The activities of the enzymes from both varieties were inhibited by KCN; however, the copper chelator diethyldithiocarbamate was inhibitory only against the C. neoformans var. gattii enzyme, as was sodium azide. The C. neoformans var. neoformans SOD was not affected by preincubation for 1 h at 70 degrees C, and it also retained most of its activity when incubated at 37 degrees C relative to its activity when incubated at 20 degrees C, in contrast to the C. neoformans var. gattii enzyme. The pronounced differences in the physical and biochemical characteristics of the Cu,Zn SODs from the two Cryptococcus varieties complement recent reports illustrating the biochemical and genetic differences between C. neoformans var. neoformans and C. neoformans var. gattii, and the successful purification of the two enzymes comprises the first step in determining what role, if any, the cryptococcal Cu,Zn SODs might have in protection against externally generated superoxide.