Ribonucleotide reductase in an anoxia tolerant vertebrate, the crucian carp

Abstract

Can enzymes that are oxygen‐dependent in all known eukaryotes, function in the anoxic crucian carp (Carassius carassius)? The crucian carp can survive for several months in anoxia, partly because of its extraordinary ability to convert lactate to ethanol, which allows it to avoid lactate accumulation. Previous studies have shown that crucian carp cells can undergo cell division in anoxia. However, DNA replication should not be possible in anoxia, because in all eukaryote organisms examined ribonucleotide reductase (RNR), which produces the building material for DNA, is dependent on oxygen to form a catalytically important radical. Therefore, the RNR in crucian carp must be exceptional in some way, possibly by forming an unusually stable radical, or by having an exceptionally high oxygen affinity. We have cloned and sequenced several RNR variants from crucian carp. Interestingly, quantitative real‐time PCR measurements in normoxic and anoxic crucian carp revealed that the expression of the small RNR subunit (R2), which is the subunit activated by oxygen, was maintained in anoxia. RNR proteins were in‐vitro expressed in E. coli for structural and enzymatic studies. Amino acid analysis and EPR spectroscopy suggest that the variants are structurally very similar to mammalian RNR, but ongoing studies will show if their oxygen affinity are different. Supported by the Research Council of Norway.