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

Abstract

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.