Isolation of the Gene for the B 12 -Dependent Ribonucleotide Reductase from Anabaena sp. Strain PCC 7120 and Expression in Escherichia coli

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Abstract
The gene for ribonucleotide reductase from Anabaena sp. strain PCC 7120 was identified and expressed in Escherichia coli . This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in E. coli , yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from E. coli extracts. Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5′-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii . In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp. strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.